Compositions and methods for targeted immunomodulatory antibodies and fusion proteins

ABSTRACT

The present invention is based on the seminal discovery that targeted immunomodulatory antibodies and fusion proteins can counter act or reverse immune tolerance of cancer cells. Cancer cells are able to escape elimination by chemotherapeutic agents or tumor-targeted antibodies via specific immunosuppressive mechanisms in the tumor microenvironment and such ability of cancer cells is recognized as immune tolerance. Such immunosuppressive mechanisms include immunosuppressive cytokines (for example, Transforming growth factor beta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloid dendritic cells (DCs). By counteracting tumor-induced immune tolerance, the present invention provides effective compositions and methods for cancer treatment, optional in combination with another existing cancer treatment. The present invention provides strategies to counteract tumor-induced immune tolerance and enhance the antitumor efficacy of chemotherapy by activating and leveraging T cell-mediated adaptive antitumor immunity against resistant or disseminated cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/231,309 filed Aug. 8, 2016, now pending, which is a continuationapplication of U.S. application Ser. No. 14/645,282 filed Mar. 11, 2015,now issued as U.S. Pat. No. 9,441,044; which is a continuationapplication of U.S. application Ser. No. 13/582,717 filed Oct. 17, 2012,now issued as U.S. Pat. No. 8,993,524; which is a 35 USC § 371 NationalStage application of International Application No. PCT/US2011/027317filed Mar. 4, 2011, now expired; which claims the benefit under 35 USC §119(e) to U.S. Application Ser. No. 61/435,671 filed Jan. 24, 2011 andto U.S. Application Ser. No. 61/311,255 filed Mar. 5, 2010, both nowexpired. The disclosure of each of the prior applications is consideredpart of and is incorporated by reference in the disclosure of thisapplication.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field of targetedimmunomodulatory antibodies and fusion proteins for cancer therapy andmore specifically to composition and methods for targetedimmunostimulatory or immunosuppressive antibodies and fusion proteins tocounteract or induce immune tolerance of cancer cells.

Background Information

The immune system provides the human body with a means to recognize anddefend itself against microorganisms and substances recognized asforeign or potentially harmful. While passive immunotherapy of cancerwith monoclonal antibodies and passive transfer of T cells to attacktumor cells have demonstrated clinical efficacy, the goal of activetherapeutic vaccination to induce these immune effectors and establishimmunological memory against tumor cells has remained challenging.Several tumor-specific and tumor-associated antigens have beenidentified, yet these antigens are generally weakly immunogenic andtumors employ diverse mechanisms to create a tolerogenic environmentthat allows them to evade immunologic attack. Strategies to overcomesuch immune tolerance and activating robust levels of antibody and/or Tcell responses hold the key to effective cancer immunotherapy.

SUMMARY OF THE INVENTION

The present invention is based on the seminal discovery that targetedimmunomodulatory antibodies and fusion proteins can counteract orreverse immune tolerance of cancer cells. Cancer cells are able toescape elimination by chemotherapeutic agents or tumor-targetedantibodies via specific immunosuppressive mechanisms in the tumormicroenvironment and such ability of cancer cells is recognized asimmune tolerance. Such immunosuppressive mechanisms includeimmunosuppressive cytokines (for example, Transforming growth factorbeta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloiddendritic cells (DCs). By counteracting tumor-induced immune tolerance,the present invention provides effective compositions and methods forcancer treatment, optional in combination with another existing cancertreatment. The present invention provides strategies to counteracttumor-induced immune tolerance and enhance the antitumor efficacy ofchemotherapy by activating and leveraging T cell-mediated adaptiveantitumor immunity against resistant or disseminated cancer cells.

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an immunomodulatory moiety. The targetingmoiety specifically binds a target molecule, and the immunomodulatorymoiety specifically binds one of the following molecules: (i)Transforming growth factor-beta (TGF-β); (ii) Programmed death-1 ligand1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2); (iii) Receptoractivator of nuclear factor-κB (RANK) ligand (RANKL); (iv) Transforminggrowth factor-beta receptor (TGF-βR); (v) Programmed death-1 (PD-1); and(vi) Receptor activator of nuclear factor-κB (RANK).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, or Fc-containing polypeptide that specifically binds acomponent of a tumor cell, tumor antigen, tumor vasculature, tumormicroenvironment, or tumor-infiltrating immune cell. In one aspect, thetargeting moiety specifically binds epidermal growth factor receptor(EGFR1, Erb-B1), HER2/neu (Erb-B2), CD20, Vascular endothelial growthfactor (VEGF), insulin-like growth factor receptor (IGF-1R),TRAIL-receptor, epithelial cell adhesion molecule, carcino-embryonicantigen, Prostate-specific membrane antigen, Mucin-1, CD30, CD33, orCD40.

In one aspect, the targeting moiety specifically binds a component of aregulatory T cell, myeloid suppressor cell, or dendritic cell. Inanother aspect, the targeting moiety specifically binds one of thefollowing molecules: (i) CD4; (ii) CD25 (IL-2α receptor; IL-2αR); (iii)cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152); (iv) Interleukin-10(IL-10); (v) Transforming growth factor-beta receptor (TGF-βR); (vi)Transforming growth factor-beta (TGF-β); (vii) Programmed Death-1(PD-1); (viii) Programmed death-1 ligand (PD-L1 or PD-L2); (ix) Receptoractivator of nuclear factor-κB (RANK); or (x) Receptor activator ofnuclear factor-κB (RANK) ligand (RANKL).

In one aspect, the immunomodulatory moiety specifically binds one of thefollowing molecules: (i) Transforming growth factor-beta (TGF-β); (ii)Programmed death-1 ligand (PD-L1 or PD-L2); or (iii) Receptor activatorof nuclear factor-κB (RANK) ligand (RANKL).

In one aspect, the immunomodulatory moiety includes a molecule thatbinds TGF-β. In another aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain of Transforming growth factor-betareceptor TGF-βRII, TGF-βRIIb, or TGF-βRIII. In another aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof TGF-βRII. In an additional aspect, the immunomodulatory moietyinhibits the activity or function of TGF-β.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain of TGF-βRII. In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Programmed Death-1(PD-1), Programmed death-1 ligand 1 (PD-L1), or Programmed death-1ligand 2 (PD-L2). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Programmed Death-1(PD-1). In an additional aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain of TGF-βRII. In another aspect, themolecule includes PD-1 ectodomain, immunoglobulin Fc region, and TGFβRIIectodomain. In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 11 or 12.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Receptor activatorof nuclear factor-κB (RANK) or Receptor activator of nuclear factor-κBligand (RANKL). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof TGF-βRII. In another aspect, the molecule includes RANK ectodomain,immunoglobulin Fc region, and TGFβRII ectodomain. In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 13or 14.

In one aspect, the immunomodulatory moiety includes a molecule thatspecifically binds to Programmed death-1 ligand 1 (PD-L1) or Programmeddeath-1 ligand 2 (PD-L2). In another aspect, the immunomodulatory moietyincludes an extracellular ligand-binding domain or ectodomain ofProgrammed Death-1 (PD-1). In an additional aspect, the immunomodulatorymoiety inhibits the activity or function of Programmed death-1 ligand 1(PD-L1).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain or ectodomain of Programmed Death-1 (PD-1). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Receptor activatorof nuclear factor-κB (RANK) or Receptor activator of nuclear factor-κBligand (RANKL). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof Programmed Death-1 (PD-1). In another aspect, the molecule includesRANK ectodomain, immunoglobulin Fc region, and PD-1 ectodomain. Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 25 or 26.

In one aspect, the immunomodulatory moiety includes a molecule thatspecifically binds to Receptor activator of nuclear factor-κB ligand(RANKL). In another aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, theimmunomodulatory moiety inhibits the activity or function of Receptoractivator of nuclear factor-κB ligand (RANKL).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain or ectodomain of Receptor activator of nuclearfactor-κB (RANK). In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35,or 36.

In one aspect, the immunomodulatory moiety includes a sequence fromProgrammed death-1 ligand 1 (PD-L1) or Programmed death-1 ligand 2(PD-L2). In an additional aspect, the immunomodulatory moiety increasesthe function of PD-1.

In one aspect, the targeting moiety specifically binds to Tumor NecrosisFactor-α (TNF-α), and the immunomodulatory moiety includes a sequencefrom Programmed death-1 ligand 1 (PD-L1) or Programmed death-1 ligand 2(PD-L2). In an additional aspect, the targeting moiety includes anantibody that binds TNF-α, and the immunomodulatory moiety includes asequence from PD-1 ligand 1 (PD-L1 or B7-H1). In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 37.In another aspect, the targeting moiety includes an extracellularligand-binding domain of tumor necrosis factor receptor 2 (TNFR2), andthe immunomodulatory moiety includes a sequence from PD-1 ligand 1(PD-L1 or B7-H1). In another aspect, the molecule includes TNFR2Extracellular ligand binding domain, immunoglobulin Fc region, and asequence from PD-L1. In another aspect, the molecule includes an aminoacid sequence corresponding to SEQ ID NO: 38 or 39.

In one aspect, the targeting moiety includes an antibody or antibodyfragment that specifically binds to CD20, CD25, or CD4, and theimmunomodulatory moiety includes a sequence from Programmed death-1ligand 1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2). In anotheraspect, the molecule includes an amino acid sequence corresponding toSEQ ID NO: 40, 41, 42, 43, 44, or 45.

In one aspect, the targeting moiety includes the extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes a sequence from Programmed death-1 ligand 1 (PD-L1) orProgrammed death-1 ligand 2 (PD-L2). In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 46 or 47.

In one aspect, the targeting moiety includes transforming growthfactor-β (TGF-β) and immunoglobulin Fc region (IgG Cγ1), and theimmunomodulatory moiety includes a sequence from Programmed death-1ligand 1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2). In anadditional aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 48 or 49.

In one aspect, the immunomodulatory moiety includes a sequence fromtransforming growth factor-β (TGF-β). In an additional aspect, theimmunomodulatory moiety activates the signaling function of transforminggrowth factor-β (TGF-β) receptor.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Tumor NecrosisFactor-α (TNF-α), and the immunomodulatory moiety includes a sequencefrom transforming growth factor-β (TGF-β). In an additional aspect, thetargeting moiety includes an antibody that binds TNF-α, and theimmunomodulatory moiety includes a sequence from TGF-β. In anotheraspect, the molecule includes an amino acid sequence corresponding toSEQ ID NO: 50. In one aspect, the targeting moiety includes anextracellular ligand-binding domain of tumor necrosis factor receptor 2(TNFR2). In another aspect, the molecule includes TNFR2 Extracellularligand binding domain, immunoglobulin Fc region, and a sequence fromtransforming growth factor-β (TGF-β). In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 51 or 52.

In one aspect, the targeting moiety includes an antibody or antibodyfragment that specifically binds to CD20, CD25 (IL-2α receptor; IL-2αR),or CD4, and the immunomodulatory moiety includes a sequence fromtransforming growth factor-β (TGF-β). In an additional aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 53,54, 55, 56, 57 or 58.

In one aspect, the targeting moiety includes an extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes a sequence from transforming growth factor-β (TGF-β). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 59 or 60.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Tumor NecrosisFactor-α (TNF-α), and the immunomodulatory moiety includes anextracellular RANKL-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, the targetingmoiety includes an antibody that binds TNF-α, and the immunomodulatorymoiety includes a sequence from an extracellular RANKL-binding domain orectodomain of RANK. In another aspect, the molecule includes an aminoacid sequence corresponding to SEQ ID NO: 61. In one aspect, thetargeting moiety includes an extracellular ligand-binding domain oftumor necrosis factor receptor 2 (TNFR2). In another aspect, themolecule includes TNFR2 Extracellular ligand binding domain,immunoglobulin Fc region, and a sequence from an extracellularRANK-binding domain or ectodomain of RANK. In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 62or 63.

In one aspect, the targeting moiety includes an extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes an extracellular RANKL-binding domain or ectodomain ofReceptor activator of nuclear factor-κB (RANK). In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 64or 65.

In one aspect, the targeting moiety includes includes a sequence fromtransforming growth factor-β (TGF-β) and immunoglobulin Fc region (IgGCγ1), and the immunomodulatory moiety includes an extracellularRANKL-binding domain or ectodomain of Receptor activator of nuclearfactor-κB (RANK). In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 66 or 67.

In one aspect, the targeting moiety includes a sequence from Programmeddeath-1 ligand 1 (PD-L1) and immunoglobulin Fc region (IgG Cγ1), and theimmunomodulatory moiety includes an extracellular RANKL-binding domainor ectodomain of Receptor activator of nuclear factor-κB (RANK). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 68 or 69.

In various aspects, the molecule is fused or directly linked to one ormore antigen, antigenic determinant, or epitope.

In another embodiment, the present invention provides a compositionincluding the molecule of the invention and a cell, wherein the cell isa tumor cell, immune cell, or dendritic cell.

In another embodiment, the present invention provides a method ofcounteracting or overcoming immune tolerance. The method includesadministering to a subject in need thereof one or more molecule of theinvention.

In another embodiment, the present invention provides a method ofpreventing or treating a neoplastic disease. The method includesadministration to a subject in need thereof one or more molecule of theinvention. In various aspects, the subject is administered one or moremolecule of the invention in combination with another anticancertherapy. In one aspect, the anticancer therapy includes achemotherapeutic molecule, antibody, small molecule kinase inhibitor,hormonal agent or cytotoxic agent. In another aspect, the anticancertherapy includes ionizing radiation, ultraviolet radiation,cryoablation, thermal ablation, or radiofrequency ablation.

In another embodiment, the present invention provides a method ofpreventing or treating a neoplastic disease. The method includesadministration to a subject in need thereof an antibody that targets anddepletes CD4+ regulatory T cells (Tregs) in combination with anothercytotoxic anticancer therapy. In one aspect, the antibody that targetsand depletes Tregs is an anti-CD4 antibody. In various aspects, thecytotoxic anticancer therapy includes a chemotherapeutic molecule,tumor-targeted antibody, small molecule kinase inhibitor, hormonal agentor tumor-targeted cytotoxic agent. In another aspect, the cytotoxicanticancer therapy includes ionizing radiation, ultraviolet radiation,cryoablation, thermal ablation, or radiofrequency ablation.

In another embodiment, the subject is administered one or more moleculeof the invention in combination with any vaccine. In another aspect, thevaccine includes a tumor antigen, tumor-associated antigen, tumorepitope, tumor antigen-containing fusion protein, tumor cell, ordendritic cell. In another aspect, the vaccine includes a pathogenantigen, pathogen-associated antigen, pathogen epitope, or pathogenantigen-containing fusion protein.

In another embodiment, the present invention provides a method fortreating immune cells wherein the cells are contacted ex vivo or invitro with a molecule of the invention. In another embodiment, thepresent invention provides a method of treatment of a neoplasticdisease. The method includes administering to a subject in need thereofa composition of immune cells contacted with a molecule of theinvention.

In another embodiment, the present invention provides a method ofinducing or promoting immune tolerance. The method includesadministering to a subject in need thereof one or more molecule of theinvention.

In another embodiment, the present invention provides a method ofpreventing or treating an autoimmune or inflammatory disease includingadministering to a subject in need thereof one or more molecule of theinvention. In one aspect, the subject is administered one or moremolecule of the invention in combination with another anti-inflammatoryor immunosuppressive therapy. In another embodiment, the presentinvention provides a method of treatment of immune cells wherein thecells are contacted ex vivo or in vitro with a molecule of theinvention. In another embodiment, the present invention provides amethod of treating an autoimmune or inflammatory disease or preventingrejection of grafted cells or tissue. The method includes administeringto a subject in need thereof a composition of immune cells contactedwith a molecule of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show exemplary amino acid sequences of transforming growthfactor beta receptor type II (TGF-β-RII) or TGF-β-RIIB or a fragmentthereof, including (i) Transforming growth factor beta receptor type II(TGF-β-RII) (SEQ ID NO: 79); and (ii) Transforming growth factor betareceptor type JIB (TGF-β-RIM) (SEQ ID NO: 80).

Also shown in FIGS. 1A-1C are exemplary truncated mutants ofTransforming growth factor beta Receptor II (TGF-β-RII) or TGF-β-RIIBincluding the Extracellular domain (ECD) region that binds TGF-β,including (i) TGF-β R-II (ΔC terminus): TGFβ RII lacking the last 38amino acids from the C-terminus (SEQ ID NO: 81) and TGF-β R-IIB (ΔCterminus): TGFβRIIB lacking the last 38 aa from the C-terminus (SEQ IDNO: 82); (ii) TGF-βR-II (Δcyt): TGFβRII lacking the kinase domain &juxtamembrane region (SEQ ID NO: 83) and TGF-βR-IIB (Δcyt): TGFβRIIBlacking the kinase domain & juxtamembrane region (SEQ ID NO: 84); (iii)TGF-β R-II containing the N-terminus region including the extracellulardomain (SEQ ID NO: 85) and TGF-β R-IIB containing the N-terminus regionincluding the extracellular domain (SEQ ID NO: 86); (iv) TGF-β R-IIcontaining the extracellular domain that binds TGF-β (SEQ ID NO: 87) andTGF-β R-IIB containing the extracellular domain that binds TGF-β (SEQ IDNO: 88); and (v) TGF-β R-II containing the region of the extracellulardomain that binds TGF-β (SEQ ID NO: 89).

In addition, FIGS. 1A-1C also show exemplary kinase-deficient mutants,deletion mutants, or point mutants of Transforming growth factor betaReceptor II (TGFβ-RII) or TGFβ-RIIB or a fragment thereof which bindsTGF-β, including (i) Transforming growth factor beta Receptor IIcontaining point mutations—amino acid sequence of TGF-β R-II (K277R)contains a point mutation in its ATP-binding site and is inactive as akinase (SEQ ID NO: 90); and (ii) Transforming growth factor betaReceptor II containing deletions in the amino acid sequence (deletionmutants)—Transforming growth factor beta Receptor II (Δi)—TGF-β R-II(Δi2) contains a deletion of amino acids 498 to 508 and is inactive as akinase (SEQ ID NO: 91).

FIG. 2 shows exemplary fusion proteins including anti-HER2/neu antibodyand Transforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-HER2/neu heavy chain+TGFβ-RII ECD fusionamino acid sequence (SEQ ID NO: 1) and anti-HER2/neu light chain aminoacid sequence (SEQ ID NO: 70).

FIG. 3 shows exemplary fusion proteins including anti-EGFR1 antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-EGFR1 heavy chain+TGFβ-RII ECD fusion aminoacid sequence (SEQ ID NO: 2) and anti-EGFR1 light chain amino acidsequence (SEQ ID NO: 71).

FIG. 4 shows exemplary fusion proteins including anti-CD20 antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-CD20 heavy chain+TGFβ-RII ECD fusion aminoacid sequence (SEQ ID NO: 3) and anti-CD20 light chain amino acidsequence (SEQ ID NO: 72).

FIG. 5 shows exemplary fusion proteins including anti-VEGF antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-VEGF heavy chain+TGFβ-RII ECD fusion aminoacid sequence (SEQ ID NO: 4) and anti-VEGF Light chain sequence (SEQ IDNO: 73).

FIG. 6 shows exemplary fusion proteins including anti-human CTLA-4antibody and Transforming growth factor-beta receptor II (TGFβ-RII)Extracellular domain (ECD), including Anti-CTLA-4 heavy chain+TGFβ-RIIExtracellular domain fusion amino acid sequence (SEQ ID NO: 5) andAnti-CTLA-4 light chain (SEQ ID NO: 74).

FIG. 7 shows exemplary fusion proteins including IL-2, Fc, andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including IL-2+Fc+TGFβ-RII Extracellular domain (SEQ IDNO: 6) and TGFβ-RII Extracellular domain+Fc+IL-2 (SEQ ID NO: 7). Thelinker GGGGSGGGGSGGGGS (SEQ ID NO: 104) is optional and can be replacedwith EPKSCDK (SEQ ID NO: 105) or another linker sequence well known inthe art. Certain amino acid sequences can be replaced in Fc, includingunderlined E with D and underlined M with L.

FIGS. 8A-8B show exemplary fusion proteins including anti-CD25 antibodyand Transforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-CD25 (Daclizumab) heavy chain and TGFβ-RIIExtracellular domain (SEQ ID NO: 8) and anti-CD25 (Daclizumab) lightchain (SEQ ID NO: 75) (FIG. 8A); and anti-CD25 (Basiliximab) heavy chainand TGFβ-RII Extracellular domain (SEQ ID NO: 9) and anti-CD25(Basiliximab) light chain (SEQ ID NO: 76).

FIG. 9 shows exemplary fusion proteins including anti-CD4 antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), including anti-CD4 heavy chain and TGFβ-RII Extracellulardomain (SEQ ID NO: 10) and anti-CD4 light chain (SEQ ID NO: 77).

FIG. 10 shows exemplary fusion proteins including Programmed Death-1(PD-1) Ectodomain, Fc, and Transforming growth factor-beta receptor II(TGFβ-RII) Extracellular domain (ectodomain), including PD-1ectodomain+Fc+TGFβRII ectodomain (SEQ ID NO: 11) and TGFβRIIectodomain+Fc+PD-1 ectodomain (SEQ ID NO: 12). The linker sequenceEPKSCDK (SEQ ID NO: 105) is optional and can be deleted or replaced withanother linker.

FIG. 11 shows exemplary fusion proteins including Receptor activator ofnuclear factor-kB (RANK) Ectodomain, Fc, and Transforming growthfactor-beta receptor II (TGFβ-RII) Extracellular domain (ectodomain),including RANK ectodomain+Fc+TGFβRII ectodomain (SEQ ID NO: 13) andTGFβRII ectodomain+Fc+RANK ectodomain (SEQ ID NO: 14). The linkersequence EPKSCDK (SEQ ID NO: 105) is optional and can be deleted orreplaced with another linker.

FIG. 12 shows exemplary immunomodulatory moiety that binds ProgrammedDeath-1 ligand 1 (PD-L1 or B7-H1) or Programmed Death-1 ligand 2 (PD-L2or B7-DC), including full-length PD-1 or fragment thereof (SEQ ID NO:92), PD-1 extracellular domain (ectodomain) or fragment thereof (SEQ IDNO: 93), and PD-1 extracellular domain (ectodomain) ligand-bindingregion (SEQ ID NO: 94).

FIG. 13 shows exemplary fusion proteins including anti-HER2/neu antibodyand PD-1 Ectodomain, including anti-HER2/neu heavy chain+PD-1 ectodomainfusion amino acid sequence (SEQ ID NO: 15) and anti-HER2/neu light chainamino acid sequence (SEQ ID NO: 70).

FIG. 14 shows exemplary fusion proteins including anti-EGFR1 antibodyand PD-1 Ectodomain, including anti-EGFR heavy chain+PD-1 ectodomainfusion amino acid sequence (SEQ ID NO: 16) and anti-EGFR light chainamino acid sequence (SEQ ID NO: 71).

FIG. 15 shows exemplary fusion proteins including anti-CD20 antibody andPD-1 Ectodomain, including anti-CD20 heavy chain+PD-1 ectodomain fusionamino acid sequence (SEQ ID NO: 17) and anti-CD20 light chain amino acidsequence (SEQ ID NO: 72).

FIG. 16 shows exemplary fusion proteins including anti-VEGF antibody andPD-1 Ectodomain, including anti-VEGF heavy chain+PD-1 ectodomain fusionamino acid sequence (SEQ ID NO: 18) and anti-VEGF Light chain sequence(SEQ ID NO: 73).

FIG. 17 shows exemplary fusion proteins including anti-human CTLA-4antibody and PD-1 Ectodomain, including anti-CTLA-4 heavy chain+PD-1ectodomain fusion amino acid sequence (SEQ ID NO: 19) and anti-CTLA-4light chain (SEQ ID NO: 74).

FIGS. 18A-18B show exemplary fusion proteins including anti-CD25antibody and PD-1 Ectodomain, including anti-CD25 (Daclizumab) heavychain and PD-1 ectodomain (SEQ ID NO: 20) and anti-CD25 (Daclizumab)light chain (SEQ ID NO: 75) (FIG. 18A), and anti-CD25 (Basiliximab)heavy chain and PD-1 ectodomain (SEQ ID NO: 21) and anti-CD25(Basiliximab) light chain (SEQ ID NO: 76) (FIG. 18B).

FIG. 19 shows exemplary fusion proteins including IL-2, Fc, and PD-1ectodomain, including IL-2+Fc+PD-1 ectodomain (SEQ ID NO: 22) and PD-1ectodomain+Fc+IL-2 (SEQ ID NO: 23). The linker GGGGSGGGGSGGGGS SEQ IDNO: 104 is optional and can be replaced with EPKSCDK SEQ ID NO: 105 oranother linker sequence well known in the art. Certain amino acidsequences can be replaced in Fc, including underlined E with D andunderlined M with L.

FIG. 20 shows exemplary fusion proteins including anti-CD4 antibody andPD-1 ectodomain, including anti-CD4 heavy chain and PD-1 ectodomain (SEQID NO: 24) and anti-CD4 light chain (SEQ ID NO: 77).

FIG. 21 shows exemplary fusion proteins including Receptor activator ofnuclear factor-kB (RANK) Ectodomain, Fc, and PD-1 ectodomain, includingRANK ectodomain+Fc+PD-1 ectodomain (SEQ ID NO: 25) and PD-1ectodomain+Fc+RANK ectodomain (SEQ ID NO: 26). The linker sequenceEPKSCDK (SEQ ID NO: 105) is optional and can be deleted or replaced withanother linker.

FIG. 22 shows exemplary immunomodulatory moiety that binds Receptoractivator of nuclear factor-kB (RANK) ligand (RANKL) includingfull-length RANK or fragment thereof (SEQ ID NO: 95), extracellularligand-binding domain or ectodomain of RANK (SEQ ID NO: 96),RANKL-binding sequences or residues of RANK (SEQ ID NO: 93), orRANKL-binding sequences of Osteoprotegerin (OPG) (SEQ ID NO: 98).

FIG. 23 shows exemplary fusion proteins including anti-HER2/neu antibodyand RANK Ectodomain, including anti-HER2/neu heavy chain+RANK ectodomainfusion amino acid sequence (SEQ ID NO: 27) and anti-HER2/neu light chainamino acid sequence (SEQ ID NO: 70).

FIG. 24 shows exemplary fusion proteins including anti-EGFR1 antibodyand RANK Ectodomain, including anti-EGFR heavy chain+RANK ectodomainfusion amino acid sequence (SEQ ID NO: 28) and anti-EGFR light chainamino acid sequence (SEQ ID NO: 71).

FIG. 25 shows exemplary fusion proteins including anti-CD20 antibody andRANK Ectodomain, including anti-CD20 heavy chain+RANK ectodomain fusionamino acid sequence (SEQ ID NO: 29) and anti-CD20 light chain amino acidsequence (SEQ ID NO: 72).

FIG. 26 shows exemplary fusion proteins including anti-VEGF antibody andRANK Ectodomain, including anti-VEGF heavy chain+RANK ectodomain fusionamino acid sequence (SEQ ID NO: 30) and anti-VEGF Light chain sequence(SEQ ID NO: 73).

FIG. 27 shows exemplary fusion proteins including anti-human CTLA-4antibody and RANK Ectodomain, including anti-CTLA-4 heavy chain+RANKectodomain fusion amino acid sequence (SEQ ID NO: 31) and anti-CTLA-4light chain (SEQ ID NO: 74).

FIGS. 28A-28B show exemplary fusion proteins including anti-CD25antibody and RANK Ectodomain, including anti-CD25 (Daclizumab) heavychain and RANK ectodomain (SEQ ID NO: 32) and anti-CD25 (Daclizumab)light chain (SEQ ID NO: 75) (FIG. 28A), and anti-CD25 (Basiliximab)heavy chain and RANK ectodomain (SEQ ID NO: 33) and anti-CD25(Basiliximab) light chain (SEQ ID NO: 76) (FIG. 28B).

FIG. 29 shows exemplary fusion proteins including IL-2, Fc, and RANKectodomain, including IL-2+Fc+RANK ectodomain (SEQ ID NO: 34) and RANKectodomain+Fc+IL-2 (SEQ ID NO: 35). The linker GGGGSGGGGSGGGGS SEQ IDNO: 104 is optional and can be replaced with EPKSCDK SEQ ID NO: 105 oranother linker sequence well known in the art. Certain amino acidsequences can be replaced in Fc, including underlined E with D andunderlined M with L.

FIG. 30 shows exemplary fusion proteins including anti-CD4 antibody andRANK ectodomain, including anti-CD4 heavy chain and RANK ectodomain (SEQID NO: 36) and anti-CD4 light chain (SEQ ID NO: 77).

FIG. 31 shows exemplary immunomodulatory moiety that binds ProgrammedDeath-1 (PD-1) including a PD-1 ligand 1 (PD-L1 or B7-H1) or PD-1 ligand2 (PD-L2 or B7-DC) or a fragment thereof (for example, SEQ ID NO: 101),full-length human PD-1 ligand 1 (B7-H1; PDCD1L1; PD-L1; or CD274)protein or a fragment thereof (SEQ ID NO: 99), and PD-L1 extracellularbinding domain (ectodomain) or fragment thereof (SEQ ID NO: 100).

FIG. 32 shows exemplary fusion proteins including anti-tumor necrosisfactor (TNFα) antibody and PD-1 ligand, including anti-TNFα heavychain+PD-1L (SEQ ID NO: 37) and anti-TNFα light chain (SEQ ID NO: 78).The sequence KKAE (SEQ ID NO: 107) can be replaced with KRVE (SEQ ID NO:108) or KKVE (SEQ ID NO: 109).

FIG. 33 shows exemplary fusion proteins including TNFR2 Extracellularligand binding domain, Fc, and PD-1 ligand, including TNFR2 ECD+IgGCγ1+PD-L1 (SEQ ID NO: 38) and PD-L1+IgG Cγ1-TNFR2 ECD (SEQ ID NO: 39).

FIG. 34 shows exemplary fusion proteins including anti-CD20 antibody andPD-1 ligand 1 (PD-L1), including anti-CD20 heavy chain+PD-L1 sequence(SEQ ID NO: 40) and anti-CD20 light chain sequence (SEQ ID NO: 72).

FIGS. 35A-35B show exemplary fusion proteins including anti-CD25antibody and PD-1 ligand 1 (PD-L1), including anti-CD25 (Daclizumab)heavy chain and PD-L1 (SEQ ID NO: 41) and anti-CD25 (Daclizumab) lightchain (SEQ ID NO: 75) (FIG. 35A), and anti-CD25 (Basiliximab) heavychain and PD-1 ectodomain (SEQ ID NO: 42) and anti-CD25 (Basiliximab)light chain (SEQ ID NO: 76) (FIG. 35B).

FIG. 36 shows exemplary fusion proteins including IL-2, Fc, and PD-1ligand 1 (PD-L1), including fusion protein hPD-1 ligand 1+Fc+IL-2 (SEQID NO: 43) and fusion protein IL-2+Fc+PD-1 ligand 1 (SEQ ID NO: 44). Thelinker GGGGSGGGGSGGGGS SEQ ID NO: 104 is optional and can be replacedwith EPKSCDK SEQ ID NO: 105 or another linker sequence well known in theart. Certain amino acid sequences can be replaced in Fc, includingunderlined E with D and underlined M with L.

FIG. 37 shows exemplary fusion proteins including anti-CD4 antibody andPD-1 ligand 1 (PD-L1), including anti-CD4 heavy chain and PD-1 ligand 1(PD-L1) (SEQ ID NO: 45) and anti-CD4 light chain (SEQ ID NO: 77).

FIG. 38 shows exemplary fusion proteins including the extracellulardomain of CTLA-4, Immunoglobulin Fc (IgG Cγ1), and a sequence from PD-1ligand (PD-L1) including Oncostatin M signal peptide+CTLA-4 ECD+IgGCγ1+PD-L1 (SEQ ID NO: 46) and PD-1L1+IgG Cγ1+CTLA-4 ECD (SEQ ID NO: 47).The IgG sequence shown can have optional C to S conversion in (boldunderlined). The linker QEPKSCDK SEQ ID NO: 110 is optional and can bereplaced with EPKSCDK SEQ ID NO: 105 or another linker sequence.

FIG. 39 shows exemplary fusion proteins including a sequence oftransforming growth factor-β (TGF-β), Immunoglobulin Fc (IgG Cγ1), and asequence of PD-1 ligand (PD-L1) including TGFβ-1+Fc+PD-L1 (SEQ ID NO:48), and PD-1L1+Fc+TGFβ-1 (SEQ ID NO: 49). The linker GGGGSGGGGSGGGGSSEQ ID NO: 104 is optional and can be replaced with EPKSCDK SEQ ID NO:105 or another linker sequence well known in the art. Certain amino acidsequences can be replaced in Fc, including underlined E with D andunderlined M with L.

FIG. 40 shows exemplary immunomodulatory moiety that binds Transforminggrowth factor-beta receptor (TGF-βR) including Transforming growthfactor-beta (TGF-β1, TGF-β2, or TGF-β3 or a fragment thereof, TGF-β1full sequence (SEQ ID NO: 102), and mature (active) TGF-β1 sequence (Ala279-Ser 390; 112 amino acids) (SEQ ID NO: 103).

FIG. 41 shows exemplary fusion proteins including an antibody that bindsTNF-α, and a sequence of transforming growth factor-β (TGF-β), includinganti-TNFα heavy chain+TGF-β1 (SEQ ID NO: 50) and anti-TNFα light chain(SEQ ID NO: 78). The sequence KKAE (SEQ ID NO: 107) can be replaced withKRVE (SEQ ID NO: 108) or KKVE (SEQ ID NO: 109).

FIG. 42 shows exemplary fusion proteins including TNFR2 Extracellularligand binding domain (TNFR2 ECD), immunoglobulin Fc (IgG Cγ1), and asequence from transforming growth factor-β (TGF-β) including TNFR2ECD+IgG Cγ1+TGF-β1 (SEQ ID NO: 51), and TGF-β1+IgG Cγ1+TNFR2 ECD (SEQ IDNO: 52).

FIG. 43 shows exemplary fusion proteins including anti-CD20 antibody anda sequence from transforming growth factor-β (TGF-β), includinganti-CD20 heavy chain+mature TGFβ1 sequence (SEQ ID NO: 53) andanti-CD20 light chain sequence (SEQ ID NO: 72).

FIGS. 44A-44B show exemplary fusion proteins including anti-CD25antibody and a sequence from transforming growth factor-β (TGF-β),including anti-CD25 (Daclizumab) heavy chain and TGF-β1 (SEQ ID NO: 54)and anti-CD25 (Daclizumab) light chain (SEQ ID NO: 75) (FIG. 44A), andanti-CD25 (Basiliximab) heavy chain and TGF-β1 (SEQ ID NO: 55) andanti-CD25 (Basiliximab) light chain (SEQ ID NO: 76) (FIG. 44B).

FIG. 45 shows exemplary fusion proteins including IL-2, Fc, and asequence from transforming growth factor-β (TGF-β), includingTGF-β1+Fc+IL-2 (SEQ ID NO: 56) and IL-2+Fc+TGF-β1 (SEQ ID NO: 57). Thelinker GGGGSGGGGSGGGGS SEQ ID NO: 104 is optional and can be replacedwith EPKSCDK SEQ ID NO: 105 or another linker sequence well known in theart. Certain amino acid sequences can be replaced in Fc, includingunderlined E with D and underlined M with L.

FIG. 46 shows exemplary fusion proteins including anti-CD4 antibody anda sequence from transforming growth factor-β (TGF-β), including anti-CD4heavy chain and TGF-β (SEQ ID NO: 58) and anti-CD4 light chain (SEQ IDNO: 77).

FIG. 47 shows exemplary Fusion proteins including the extracellulardomain of CTLA-4, Immunoglobulin Fc (IgG Cγ1), and a sequence fromtransforming growth factor-β (TGF-β) including Oncostatin M signalpeptide+CTLA-4 ECD+IgG Cγ1+TGF-β1 (SEQ ID NO: 59), and TGF-β1+IgGCγ1+CTLA-4 ECD (SEQ ID NO: 60). The IgG sequence shown can have optionalC to S conversion in (bold underlined). The linker QEPKSCDK SEQ ID NO:110 is optional and can be replaced with EPKSCDK SEQ ID NO: 105 oranother linker sequence.

FIG. 48 shows exemplary fusion proteins including an antibody that bindsTNF-α, and a sequence of RANK ectodomain, including anti-TNFα heavychain+RANK ectodomain (SEQ ID NO: 61) and anti-TNFα light chain (SEQ IDNO: 78). The sequence KKAE (SEQ ID NO: 107) can be replaced with KRVE(SEQ ID NO: 108) or KKVE (SEQ ID NO: 109).

FIG. 49 shows exemplary fusion proteins including TNFR2 Extracellularligand binding domain (TNFR2 ECD), immunoglobulin Fc (IgG Cγ1), and asequence from RANK ectodomain including TNFR2 ECD+IgG Cγ1+RANKectodomain (SEQ ID NO: 62), and RANK ectodomain+IgG Cγ1+TNFR2 ECD (SEQID NO: 63).

FIG. 50 shows exemplary Fusion proteins including the extracellulardomain of CTLA-4, Immunoglobulin Fc (IgG Cγ1), and a sequence from RANKectodomain including Oncostatin M signal peptide+CTLA-4 ECD+IgG Cγ1+RANKectodomain (SEQ ID NO: 64), and RANK ectodomain+IgG Cγ1+CTLA-4 ECD (SEQID NO: 65). The IgG sequence shown can have optional C to S conversionin (bold underlined). The linker QEPKSCDK SEQ ID NO: 110 is optional andcan be replaced with EPKSCDK SEQ ID NO: 105 or another linker sequence.

FIG. 51 shows exemplary fusion proteins including a sequence fromtransforming growth factor-β (TGF-β), immunoglobulin Fc (IgG Cγ1), and asequence from RANK ectodomain including TGF-β+IgG Cγ1+RANK ectodomain(SEQ ID NO: 66), and RANK ectodomain+IgG Cγ1+TGF-β (SEQ ID NO: 67).

FIG. 52 shows exemplary fusion proteins including a sequence from PD-1ligand (PD-L1), immunoglobulin Fc (IgG Cγ1), and a sequence from RANKectodomain including PD-L1+IgG Cγ1+RANK ectodomain (SEQ ID NO: 68), andRANK ectodomain+IgG Cγ1+PD-L1 (SEQ ID NO: 69).

FIGS. 53A-53G show Regulatory T cells (Treg) accumulate in the tumormicroenvironment and counteract the ability of chemotherapy to activateCD8⁺ T cell-mediated antitumor immunity. (FIG. 53A) Surface exposure ofcalreticulin (CRT) in response to treatment of hum^(a)n (SW780) andmurine (MB49) cancer cells with doxorubicin (10 μM) for 4 h. The surfaceexposure of CRT was determined by immunofluorescence cytometry ofuntreated control or doxorubicin-treated cells stained withDylight-labeled anti-CRT antibody or an isotype control (IgG1) antibody.(FIG. 53B) Priming of tumor-reactive immune responses by MB49 tumorcells treated with doxorubicin ex vivo or in vivo. 5×10⁶ MB49 cells thatwere pre-treated ex vivo with doxorubicin (10 μM) for 4 h were injectedinto one flank of syngeneic immunocompetent C57BL/6 mice. Alternatively,C57BL/6 mice were injected with 5×10⁵ live MB49 tumor cells and thenadministered intratumoral doxorubicin (10 μg) at 10 d following tumorinoculatio^(n). Tumor-reactive immune responses were determined bymeasuring production of IFN-γ by draining lymph node (DLN) cells inresponse to in vitro re-challenge with either MB49 cell lysates, anirrelevant peptide (Hemagglutinin-HA), or medium alone. (FIG. 53C)Vaccination with doxorubicin-treated tumor cells induces CD8⁺ Tcell-mediated antitumor immunity that prevents tumor formation followingre-challenge with live tumor cells. MB49 cells (5×10⁶) that werepre-treated in vitro with doxorubicin (10 μM) for 4 h were injectedsubcutaneously into one flank of syngeneic immunocompetent C57BL/6 mice.Nave or vaccinated mice were challenged with untreated live MB49 tumorcells injected into the opposite flank with or without pre-treatmentwith an anti-CD8 antibody (Clone GK2.43)(5 μg×2 doses, iv) to depleteCD8⁺ T cells. (FIG. 53D) Delayed administration of chemotherapy in micewith pre-established tumors decreases its immunogenicity and antitumorefficacy. C57BL/6 mice were injected with 5×10⁵ live syngeneic MB49tumor cells and then administered intratumoral doxorubicin (10 μg) atd3, d7, or d10 foll^(owi)ng tumor inoculation. (FIG. 53E) Tumors fosterthe accumulation of CD4⁺CD25⁺FoxP3⁺ cells (Tregs) in theirmicroenvironment. Flow cytometric analyses of the percentage ofCD4⁺CD25⁺FoxP3⁺ c^(e)lls (Tregs) among CD4⁺ T lymphocytes isolated fromthe spleen, draining lymph nodes (DLN), and tumors of immunocompetentC57BL/6 mice at d0 and d14 after subcutaneous inoculation of 5×10⁵ liveMB49 tumor cells. (FIG. 53F) Tregs infiltrating the tumormicroenvironment suppress priming of tumor-reactive immune responses bydoxorubicin-treated tumor cells. Naïve C57BL/6 mice were vaccinated with5×10⁶ doxorubicin-killed MB49 cells with or without intravenous adoptivetransfer of 5×10⁶ CD4⁺CD25⁺ cells isolated from tumors and DLN oftumor-bearing mice via immunomagnetic separation. Tumor-reactive immuneresponses were determined by measuring production of IFN-γ by draininglymph node (DLN) cells in response to in vitro re-challenge with eitherMB49 cell lysates, an irrelevant peptide (Hemagglutinin-HA), or mediumalone. (FIG. 53G) Tregs infiltrating the tumor microenvironment suppressthe activation of adaptive antitumor immunity in response tochemotherapy-induced tumor cell death. Naïve C57BL/6 mice werevaccinated with 5×10⁶ doxorubicin-killed MB49 cells (left flank) with orwithout pre-treatment with either an anti-CD8 antibody (Clo^(ne)GK2.43)(5 μg×2 doses, iv) to deplete CD8⁺ T cells or adoptive transferof 5×10⁶CD4⁺CD25⁺ cells isolated from tumors and DLN of tumor-bearingmice. Protective antitumor immunity in vaccinated mice was determined byassessment of tumor growth upon challenge with untreated live MB49 tumorcells injected into the opposite flank.

FIGS. 54A-54F show inhibition of TGF-β in the tumor microenvironmentreduces ‘adaptive’ FoxP3⁺ regulatory T cells and enhances the antitumorefficacy chemotherapy. (FIG. 54A) Tumor growth results in a progressiveincrease in the level of serum TGF-β. Levels of TGF-β in serum of miceat d0, d14, and d28 following inoculation of 5×10⁵ live MB49 tumor cellswere evaluated utilizing ELISA. (FIG. 54B) Tumor cell-autonomousexpression of TGF-β is the dominant source of elevated TGF-β intumor-bearing mice. Tumor cells or draining lymph node cells isolatedfrom either tumor-bearing mice or their tumor-free counterparts werecultured ex vivo in serum-free medium for 24 h and the amount ofTGF-β/10⁶ cells in supernatants was measured by ELISA. (FIG. 54C)TGFβRII:Fc sequesters TGF-β in supernatants of MB49 tumor cells in aconcentration-dependent manner. MB49 tumor cells were cultured in thepresence of graded concentrations of TGFβRII:Fc (0-400 ng/ml) for 24 hfollowed by measurement of TGF-β (pg/ml/10⁶ cells) in supernatants viaELISA. (FIG. 54D) TGF-β induces ‘adaptive’ FoxP3⁺ regulatory T cells inthe tumor microenvironment. At 5 d following inoculation of MB49 tumorcells, mice were either left untreated (control) or treated withTGFβRII:Fc (1 μg intratumoral; twice weekly) for 3 weeks followed byflow cytometric analyses of intracellular FoxP3 expression in CD4⁺CD25⁺T cells infiltrating the tumors. (FIGS. 54E, 54F). Sequestration ofintratumoral TGF-β with TGFβRII:Fc reduces CD4⁺CD25⁺FoxP3⁺ Tregs intumor tissue and improves the antitumor efficacy of doxorubicin. MB49tumor-bearing mice were administered doxorubicin (5 mg/kg i.p. weekly×3)with or without twice weekly treatment with TGFβRII:Fc (1 μgintratumoral). The percentage of CD4⁺CD25⁺FoxP3⁺ cells (Tregs) amongtumor cells was assessed by flow cytometry (FIG. 54E), and tumor volumewas monitored to determine the effect of counteracting tumor-inducedTGF-β-mediated immune tolerance on the in vivo antitumor efficacy ofdoxorubicin (FIG. 54F).

FIGS. 55A-55D show that anti-CD4 antibody-mediated depletion of CD4⁺regulatory T cells facilitates chemotherapy-induced activation oftumor-reactive CD8⁺ T cells and enhances the antitumor efficacy ofchemotherapy. (FIG. 55A) In vivo depletion of tumor-infiltratingCD4⁺CD25⁺FoxP3⁺ T cells by treatment of tumor-bearing mice with anti-CD4antibody. C57BL/6 mice injected with 5×10⁵ MB49 tumor cells s.c. wereleft untreated (control) or administered an anti-CD4 antibody (CloneGK1.5) i.p. at 5 d and 9 d following tumor challenge. CD4⁺CD25⁺FoxP3⁺ Tcells infiltrating tumors isolated from mice at d16 following tumorchallenge were detected by flow cytometry. (FIG. 55B) Target-specificdepletion of either CD4⁺ T cells, CD4⁺CD25⁺FoxP3⁺ T cells, or CD8⁺ Tcells by treatment of tumor-bearing mice with anti-CD4 antibody oranti-CD8 antibody. C57BL/6 mice injected s.c. with 5×10⁵ MB49 tumorcells were left untreated or treated with doxorubicin (5 mg/kg i.p.weekly×3) beginning at d7 following tumor inoculation, with or withoutadministration of either anti-CD4 antibody (Clone GK1.5) or anti-CD8antibody (Clone GK2.43) at d5 and d9 following tumor inoculation. Flowcytometric analyses of peripheral blood mononuclear cells isolated frommice at d16 following tumor challenge determined the percentage of CD4⁺T cells or CD8⁺ T cells among total mononuclear cells, and thepercentage of CD4⁺CD25⁺FoxP3⁺ T cells among total CD4⁺ T cells. (FIG.55C) Depletion of CD4⁺ regulatory T cells facilitateschemotherapy-induced activation of tumor-reactive CD8⁺ T cells. C57BL/6mice injected s.c. with 5×10⁵ MB49 tumor cells were left untreated ortreated with doxorubicin (5 mg/kg i.p. weekly×3) beginning at d7following tumor inoculation, with or without administration of anti-CD4antibody (Clone GK1.5) at d5 and d9 following tumor inoculation.Tumor-reactive immune responses were determined by flow cytometricanalyses of IFN-γ expression in CD8⁺ T cells from the tumor and draininglymph node in response to in vitro stimulation with MB49 cell lysates.(FIG. 55D) Depletion of CD4⁺ regulatory T cells augments the in vivoantitumor efficacy of chemotherapy via activation of tumor-reactive CD8⁺T cells. C57BL/6 mice injected s.c. with 5×10⁵ MB49 tumor cells wereleft untreated or treated with doxorubicin (5 mg/kg i.p. weekly×3)beginning at d7 following tumor inoculation, with or withoutadministration of either anti-CD4 antibody (Clone GK1.5) or anti-CD8antibody (Clone GK2.43) at d5 and d9 following tumor inoculation. Tumorvolume was monitored to determine the effect of depleting either CD4⁺ Tcells or CD8⁺ T cells on the in vivo antitumor efficacy of doxorubicin.

FIGS. 56A-56F show anti-CD4 antibody-mediated depletion of CD4⁺regulatory T cell's augments and sustains the antitumor effect ofchemotherapy by enabling activation of adaptive antitumor immunity.(FIG. 56A) Surface exposure of calreticulin (CRT) in response totreatment of MB49 cancer cells with either cisplatin or the combinationof cisplatin and gemcitabine for 4 h. The surface exposure of CRT wasdetermined by immunofluorescence cytometry of untreated control orchemotherapy-treated cells stained with Dylight-labeled anti-CRTantibody or an isotype control (IgG1) antibody. (FIGS. 56B, 56C)Depletion of CD4⁺ regulatory T cells enables cisplatin-inducedactivation of tumor-reactive IFN-γ⁺CD8⁺ T cells and effector memory(CD8⁺CD62L⁻) T cells. C57BL/6 mice injected s.c. with 5×10⁵ MB49 tumorcells were left untreated or treated with cisplatin (0.5 mg/kg i.p.weekly×4) beginning at d7 following tumor inoculation, with or withoutadministration of anti-CD4 antibody (Clone GK1.5) at d5 and d9 followingtumor inoculation. Tumor-reactive immune responses were determined byflow cytometric analyses of IFN-γ expression in CD8⁺ T cells from thetumor and draining lymph node (DLN) in response to in vitro stimulationwith MB49 cell lysates (FIG. 56B). The percentage of effector memoryT_(EM) cells was determined by flow cytometric analyses of CD8⁺CD62L⁻cells (FIG. 56C). (FIGS. 56D, 56E and 56F) Depletion of CD4⁺ regulatoryT cells augments the in vivo antitumor efficacy of chemotherapy viaactivation of tumor-reactive CD8⁺ T cells. C57BL/6 mice injected s.c.with 5×10⁵ MB49 tumor cells were left untreated or treated with eithercisplatin (0.5 mg/kg) or the combination of cisplatin and gemcitabine(i.p. weekly×4) beginning at d7 following tumor inoculation, with orwithout administration of either anti-CD4 antibody (Clone GK1.5) oranti-CD8 antibody (Clone GK2.43) at d5 and d9 following tumorinoculation. Tumor volume was monitored to determine the effect ofdepleting either CD4⁺ T cells or CD8⁺ T cells on the in vivo antitumorefficacy of chemotherapy and the percentage of mice exhibiting completetumor-regression by d50 following tumor inoculation. Establishment ofadaptive antitumor immunity following regression of primary tumors wasdetermined by re-challenging mice with live MB49 tumor cells in theopposite flank.

FIGS. 57A-57H show that Chemotherapy-induced expression of NKG2D ligandson tumor cells cooperates with depletion of CD4⁺ regulatory T cells tostimulate CD8⁺ T cell-mediated tumor regression. (FIG. 57A) Genotoxicchemotherapeutic agents induce expression of mouse NKG2D ligands (Rae-1)on cancer cells. Kinetics of the upregulation of Rae1 transcripts inmouse CT26 colon cancer cells was determined by quantitative real-timePCR following treatment with irinotecan (25 μg/ml) or oxaliplatin (10μg/ml). Quantitative RT-PCR was carried out using Rae-1 specific primers[sense, 5′-CTAGTGCCACCTGGGAATTCA-3′ (SEQ ID NO: 111); anti-sense,5′-CATCATTAGCTGATCTCCAGCTCA-3′(SEQ ID NO: 112)] and probe[5′-6-FAM-CATCAGTGACAGTTACTTCTTCACCTTCTACACAGAGA-Tamra-3′ (SEQ ID NO:113)]. (FIG. 57B) Genotoxic chemotherapeutic agents inducep53-independent cell surface expression of human NKG2D ligands(MHC-I-related A and B molecules—MICA/MICB) on cancer cells. Isogenicp53-proficient (p53^(+/+)) or p53-deficient (p53^(−/−)) HCT116 cellswere treated with irinotecan (25 μg/ml) for 16 h or left untreated.Irinotecan-induced upregulation of cell surface expression of MICA/B wasdetermined by flow cytometryic analysis of tumor cells labeled with ananti-human MICA/B MAb (R&D Systems). (FIG. 57C) and (FIG. 57D) Inductionof NKG2D ligands contributes to the antitumor effect of chemotherapy invivo. Immunocompetent Balb/C mice injected s.c. with 2×10⁵ syngeneicC126 tumor cells were treated with irinotecan (50 mg/kg i.p weekly×3)beginning at d5 following tumor inoculation, with or withoutpre-treatment with an NKG2D blocking antibody (CXS, eBIOscience) (200 μgi.p.) at 16 h before each dose of chemotherapy. Tumor volume wasmonitored to determine the effect of NKG2D blockade on the in vivoantitumor efficacy of irinotecan. (FIG. 57E) In vivo depletion ofCD4⁺CD25⁺FoxP3⁺ T cells by treatment of tumor-bearing mice with anti-CD4antibody. Balb/C mice injected with 2×10⁵ CT26 tumor cells s.c. wereleft untreated or treated with irinotecan (50 mg/kg i.p weekly×3)beginning at d7 following tumor inoculation, with or withoutadministration of anti-CD4 antibody (Clone GK1.5) at d5 and d9 followingtumor inoculation. CD4⁺CD25⁺FoxP3⁺ T cells in spleen and draining lymphnode isolated from mice at d16 following tumor challenge were detectedby flow cytometry. (FIG. 57F) Depletion of CD4⁺ regulatory T cellsfacilitates irinotecan-induced activation of tumor-reactive IFN-γ⁺CD8⁺ Tcells. Balb/C mice injected with 2×10⁵ C126 tumor cells s.c. were leftuntreated or treated with irinotecan (50 mg/kg i.p weekly×3) beginningat d7 following tumor inoculation, with or without administration ofanti-CD4 antibody (Clone GK1.5) at d5 and d9 following tumorinoculation. Tumor-reactive immune responses were determined by flowcytometric analyses of IFN-γ expression in CD8⁺ T cells from the tumorand draining lymph node (DLN) in response to in vitro stimulation witheither CT26 cell lysates, an irrelevant peptide (Hemagglutinin-HA), ormedium alone. (FIG. 57G) and (FIG. 57H) Chemotherapy-induced expressionof NKG2D ligands on tumor cells cooperates with depletion of CD4⁺regulatory T cells to stimulate CD8⁺ T cell-mediated tumor regression.Balb/C mice injected with 2×10⁵ CT26 tumor cells s.c. were leftuntreated or treated with irinotecan (50 mg/kg i.p weekly×3) beginningat d7 following tumor inoculation, with or without administration ofanti-CD4 antibody (Clone GK1.5) and/or anti-CD8 antibody (Clone GK2.43)at d5 and d9 following tumor inoculation. Tumor volume was monitored todetermine the effect of depleting CD4⁺ T cells and/or CD8⁺ T cells onthe in vivo antitumor efficacy of irinotecan.

DETAILED DESCRIPTION OF THE INVENTION

Targeted immunostimulatory antibodies and/or fusion proteins forprevention or treatment of cancer: Chemotherapy is a cornerstone ofsystemic treatment of patients with most common types of advancedcancers. The vast majority of human cancers harbor genetic alterationsand signaling mechanisms that impair the direct death signaling pathwaysentrained by chemotherapeutic agents. Although chemotherapeutic agentsemploy diverse mechanisms to directly kill tumor cells, the presentinvention provides that these agents have immuno-adjuvant effects whichactivate innate and adaptive antitumor immune responses that are crucialfor their antitumor efficacy in vivo. The present invention alsoprovides that antitumor CD8⁺ T cells play an instrumental role in the invivo response of tumors to diverse cytotoxic chemotherapeutic agents.Although chemotherapeutic agents can induce “immunogenic” tumor celldeath and facilitate cross-presentation of antigens by dendritic cells,tumors create a tolerogenic environment that allows them to suppress theactivation of innate and adaptive immune responses and evade immunologicattack by immune effector cells. The present invention provides thatstrategies to counteract tumor-induced immune tolerance in the tumormicroenvironment can enhance the antitumor efficacy of chemotherapy byactivating and leveraging T cell-mediated adaptive antitumor immunityagainst disseminated cancer cells.

The present invention is based on the seminal discovery that targetedimmunomodulatory antibodies and fusion proteins can counteract orreverse immune tolerance of cancer cells. Cancer cells are able toescape elimination by chemotherapeutic agents or tumor-targetedantibodies via specific immunosuppressive mechanisms in the tumormicroenvironment and such ability of cancer cells is recognized asimmune tolerance. By counteracting tumor-induced immune tolerance, thepresent invention provides effective compositions and methods for cancertreatment, optional in combination with another existing cancertreatment.

The present invention provides compositions and methods for targetedimmunostimulatory antibodies and fusion proteins that counteract immunetolerance in the tumor microenvironment and promote T cell-mediatedadaptive antitumor immunity for maintenance of durable long-termprotection against recurrent or disseminated cancers. Thesetumor-targeted immunostimulatory molecules are designed to facilitateeffective long term T cell-mediated immune responses against tumor cellsby at least one of the following:

(i) promoting death of tumor cells via enhancement of antibody-dependentcellular cytotoxicity (ADCC);

(ii) facilitating effective cross-presentation of tumor antigen(s) fromdying tumor cells by augmenting maturation of dendritic cells (DCs); and

(iii) increasing activation and proliferation of antitumor CD8+ T cellsby negating immune suppression mediated by regulatory T cells andmyeloid suppressor cells. These antitumor immune responses may beactivated in tandem with the sensitization of tumor cells to immuneeffector-mediated cytotoxicity, thereby establishing a positive feedbackloop that augments tumor cytoreduction and reinforces adaptive antitumorimmunity. The tumor-targeted immunostimulatory monoclonal antibodies(mAbs) of the present invention provides the ability to generate andboost antitumor immunity to multiple cross-presented tumor antigensobtained from endogenous tumor cells during the course of therapy (as anin situ tumor vaccine), while simultaneously leveraging the antitumorimmune response to eliminate disseminated cancer cells. Accordingly, thetargeted immunostimulatory antibodies and fusion proteins of theinvention can integrate the hitherto distinct fields of passive andactive immunotherapy and provide a novel platform for simultaneouslyleveraging the synergistic benefits of these strategies to entraineffective innate and adaptive immune responses against targeted cancers.

While passive immunotherapy of cancer with tumor-targeted monoclonalantibodies has demonstrated clinical efficacy, the goal of activetherapeutic vaccination to induce T cell-mediated immunity and establishimmunological memory against tumor cells has remained challenging.Several tumor-specific and tumor-associated antigens have beenidentified, yet tumors employ diverse mechanisms to create a tolerogenicenvironment that allows them to suppress the activation of a Tcell-mediated antitumor immune response. The tumor-targetedimmunostimulatory antibodies and/or fusion proteins of the invention aredesigned to overcome such immune tolerance in the tumor microenvironmentand activate robust levels of T cell responses for effective cancerimmunotherapy or chemo-immunotherapy. Accordingly, the tumor-targetedimmunostimulatory antibodies and/or fusion proteins of the inventionhave broad clinical relevance for advancing the treatment of many typesof human cancers.

The tumor-targeted immunostimulatory mAbs and/or fusion proteins of theinvention provide their ability to generate and boost antitumor immunityto multiple cross-presented tumor antigens obtained from endogenoustumor cells during the course of therapy (as an in situ tumor vaccine),while simultaneously leveraging the antitumor immune response toeliminate disseminated cancer cells. Accordingly, the tumor-targetedimmunostimulatory antibodies and/or fusion proteins of the invention canintegrate the hitherto distinct fields of passive and activeimmunotherapy and provide a novel platform for simultaneously leveragingthe synergistic benefits of these strategies to entrain effective innateand adaptive immune responses against targeted cancers. This approach ofthe present invention is distinguished from and superior to conventionaltumor antigen-, allogeneic tumor cell- or DC-based vaccines in at leastone of the following aspects: (i) There is no a priori requirement todefine, clone and purify individual tumor antigens, since the patient'stumor itself is the in vivo source of antigens; (ii) Multivalentantitumor immune responses that are naturally tailored against antigensfrom the patient's own tumor are less likely to allow immune escape thana pre-selected tumor antigen; (iii) The activation of antitumor immuneresponses by the immuno-adjuvant effects of tumor-targetedimmunostimulatory antibodies or fusion proteins occurs in tandem withthe sensitization of tumor cells to immune effector-mediatedcytotoxicity, thereby establishing a positive feedback loop thataugments tumor cytoreduction and reinforces adaptive antitumor immunity;and (iv) The molecules of the invention have broad clinical relevancefor advancing the treatment of many types of human cancers.

In addition, the targeted immunostimulatory antibodies and/or fusionproteins of the invention are distinguished from and superior toexisting therapeutic molecules in at least one of the following aspects:(i) to counteract immune tolerance in the tumor microenvironment andpromote T cell-mediated adaptive antitumor immunity for maintenance oflong-term protection against recurrent or disseminated cancers (forprevention or treatment of diverse cancers); (ii) to produce immune cellcompositions for adoptive cellular therapy of diverse cancers; and (iii)to serve as immune adjuvants or vaccines for prophylaxis of diversecancers or infectious diseases.

The targeted immunostimulatory antibodies and/or fusion proteins of theinvention provide the ability to disrupt immunosuppressive networks inthe tumor microenvironment. Tumors employ a wide array of regulatorymechanisms to avoid or suppress the immune response. Cancer cellsactively promote immune tolerance in the tumor microenvironment via theexpression of cytokines and molecules that inhibit the differentiationand maturation of antigen-presenting dendritic cells. Theimmunosuppressive cytokines and ligands produced by tumor cells includethe following: (i) Transforming growth factor-beta (TGF-β); (ii)Programmed death-1 ligand 1 (PD-L1; B7-H1); (iii) Vascular endothelialgrowth factor (VEGF); and (iv) Interleukin-10 (IL-10). In addition toblocking dendritic cell (DC) maturation, these molecules promote thedevelopment of specialized subsets of immunosuppressive CD4⁺ T cells(regulatory T cells; Treg cells) and myeloid-derived suppressor cells(MDSC). Tregs are a minority sub-population of CD4⁺ T cells thatconstitutively express CD25 [the interleukin-2 (IL-2) receptor α-chain]and the forkhead box P3 (FOXP3) transcription factor. Tregs(CD4+CD25+FoxP3+ cells) maintain immune tolerance by restraining theactivation, proliferation, and effector functions of a wide range ofimmune cells, including CD4⁺ and CD8⁺ T cells, natural killer (NK) andNKT cells, B cells and antigen presenting cells (APCs) in vitro and invivo. The accumulation of Treg cells in the tumor microenvironmentre-inforces tumor immune tolerance and facilitates tumor progression andmetastases. The increased expression of immunosuppressive cytokines(TGF-β; PD-L1) and tumor-infiltrating Tregs is correlated with areduction of survival of patients with diverse types of cancers. Thepresent invention provides that tumor-induced immune tolerance mediatedvia Tregs is a crucial determinant of the resistance of cancers tocytotoxic chemotherapeutic agents and tumor-targeted antibodies. Thetargeted immunostimulatory antibodies and/or fusion proteins of theinvention inhibit key immunosuppressive molecules expressed by thetargeted tumor cell or tumor-infiltrating Treg cells and myeloidsuppressor cells (DCs or MDSC). As such, they provide the targetedability to inhibit the development or function of Tregs within the tumormicroenvironment. In another aspect, they provide the ability tocounteract Treg-induced immune suppression in the tumormicroenvironment.

The targeted immunostimulatory antibodies and/or fusion proteins of theinvention provide the ability to inhibit the development or function ofTregs and myeloid suppressor cells (DCs or MDSC) within the tumormicroenvironment. Tregs (CD4+CD25+FoxP3+ cells) express an array ofimmunosuppressive cytokines and molecules which act in concert to induceimmune tolerance and promote tumor progression and metastases. Theseinclude: (i) Cytotoxic T-lymphocyte associated protein 4 (CTLA-4;CD152), a co-inhibitory receptor that binds to the ligands CD80 (B7-1)or CD86 (B7-2) on the antigen presenting cell (APC) and inhibitsco-stimulation of T cells; (ii) Programmed death-1 ligand 1 (PD-L1;B7-H1), a ligand which engages the co-inhibitory receptor Programmeddeath-1 (PD-1) and inhibits T cell activation and proliferation. (iii)Transforming growth factor-beta (TGF-β), a cytokine which regulatesimmune responses by restricting the maturation and antigen-presentingfunction of dendritic cells, inhibiting the proliferation and activationof naïve T cells, suppressing the expression of cytotoxic molecules(Granzyme A/B, FasL, Apo2L/TRAIL, IFN-γ) in immune effector cells, andpromoting the development and function of Tregs; (iv) Receptor activatorof nuclear factor-α ligand (RANKL), a ligand which engages Receptoractivator of nuclear factor-κB (RANK) and promotes osteoclastdifferentiation, Treg development, and tumor metastases. In addition,Tregs express other surface molecules; (v) LAG-3, a CD4-related moleculethat binds MHC class II; (vi) glucocorticoid-induced tumor necrosisfactor receptor family-related gene (GITR; TNFRSF18); and (vii) IL-10.The targeted immunostimulatory antibodies and/or fusion proteins of theinvention provide the ability to bind a targeted molecule expressed byTregs or myeloid suppressor cells while concurrently sequestering andinhibiting one or more immunosuppressive molecule that promotes theirdevelopment, survival or function. In one aspect, the targetedimmunostimulatory antibodies and/or fusion proteins directly deplete thenumber of Tregs.

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an immunomodulatory moiety. The targetingmoiety specifically binds a target molecule on the tumor cell or tumormicroenvironment (tumor stroma, tumor vasculature, or tumor infiltratingimmune cell), and the immunomodulatory moiety specifically binds animmunosuppressive molecule expressed by the targeted tumor cell ortumor-infiltrating Treg cells and myeloid suppressor cells (DC or MDSC).

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an immunomodulatory moiety. The targetingmoiety specifically binds a target molecule expressed by Treg cells,myeloid suppressor cells (MDSC), or dendritic cells (DC), and theimmunomodulatory moiety specifically binds an immunosuppressive moleculethat promotes their development, survival or function.

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an immunomodulatory moiety. The targetingmoiety specifically binds a target molecule, and the immunomodulatorymoiety specifically binds one of the following molecules: (i)Transforming growth factor-beta (TGF-β); (ii) Programmed death-1 ligand1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2); (iii) Receptoractivator of nuclear factor-κB (RANK) ligand (RANKL); (iv) Vascularendothelial growth factor (VEGF); (v) Transforming growth factor-betareceptor (TGF-βR); (vi) Programmed death-1 (PD-1); and (vii) Receptoractivator of nuclear factor-κB (RANK).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, or Fc-containing polypeptide that specifically binds acomponent of a tumor cell, tumor antigen, tumor vasculature, tumormicroenvironment, or tumor-infiltrating immune cell. In one aspect, thetargeting moiety specifically binds epidermal growth factor receptor(EGFR1, Erb-B1), HER2/neu (Erb-B2), CD20, Vascular endothelial growthfactor (VEGF), insulin-like growth factor receptor (IGF-1R),TRAIL-receptor, epithelial cell adhesion molecule, carcino-embryonicantigen, Prostate-specific membrane antigen, Mucin-1, CD30, CD33, orCD40.

In one aspect, the targeting moiety specifically binds a component of aregulatory T cell, myeloid suppressor cell, or dendritic cell. Inanother aspect, the targeting moiety specifically binds one of thefollowing molecules: (i) CD4; (ii) CD25 (IL-2α receptor; IL-2αR); (iii)cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152); (iv) Interleukin-10(IL-10); (v) Transforming growth factor-beta receptor (TGF-βR); (vi)Transforming growth factor-beta (TGF-β); (vii) Programmed Death-1(PD-1); (viii) Programmed death-1 ligand (PD-L1 or PD-L2); (ix) Receptoractivator of nuclear factor-κB (RANK); (x) Receptor activator of nuclearfactor-κB (RANK) ligand (RANKL); (xi) LAG-3; or (xii)glucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; TNFRSF18).

In one aspect, the immunomodulatory moiety specifically binds one of thefollowing molecules: (i) Transforming growth factor-beta (TGF-β); (ii)Programmed death-1 ligand (PD-L1 or PD-L2); (iii) Receptor activator ofnuclear factor-α (RANK) ligand (RANKL); or (iv) vascular endothelialgrowth factor (VEGF).

In one aspect, the immunomodulatory moiety includes a molecule thatbinds TGF-β. In another aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain of Transforming growth factor-betareceptor TGF-βRII, TGF-βRIIb, or TGF-βRIII. In another aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof TGF-βRII. In another aspect, the molecule includes a TGF-β-bindingamino acid sequence corresponding to SEQ ID NOs: 79-91. In an additionalaspect, the immunomodulatory moiety inhibits the activity or function ofTGF-β.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain of TGF-βRII. In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Programmed Death-1(PD-1), Programmed death-1 ligand 1 (PD-L1), or Programmed death-1ligand 2 (PD-L2). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Programmed Death-1(PD-1). In an additional aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain of TGF-βRII. In another aspect, themolecule includes PD-1 ectodomain, immunoglobulin Fc region, and TGFβRIIectodomain. In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 11 or 12.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Receptor activatorof nuclear factor-κB (RANK) or Receptor activator of nuclear factor-κBligand (RANKL). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof TGF-βRII. In another aspect, the molecule includes RANK ectodomain,immunoglobulin Fc region, and TGFβRII ectodomain. In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 13or 14.

In one aspect, the immunomodulatory moiety includes a molecule thatspecifically binds to Programmed death-1 ligand 1 (PD-L1) or Programmeddeath-1 ligand 2 (PD-L2). In another aspect, the immunomodulatory moietyincludes an extracellular ligand-binding domain or ectodomain ofProgrammed Death-1 (PD-1). In another aspect, the molecule includes aPD-L1-binding amino acid sequence corresponding to SEQ ID NO: 92, 93, or94. In an additional aspect, the immunomodulatory moiety inhibits theactivity or function of Programmed death-1 ligand 1 (PD-L1).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain or ectodomain of Programmed Death-1 (PD-1). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Receptor activatorof nuclear factor-κB (RANK) or Receptor activator of nuclear factor-κBligand (RANKL). In another aspect, the targeting moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In an additional aspect, theimmunomodulatory moiety includes an extracellular ligand-binding domainof Programmed Death-1 (PD-1). In another aspect, the molecule includesRANK ectodomain, immunoglobulin Fc region, and PD-1 ectodomain. Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 25 or 26.

In one aspect, the immunomodulatory moiety includes a molecule thatspecifically binds to Receptor activator of nuclear factor-κB ligand(RANKL). In another aspect, the immunomodulatory moiety includes anextracellular ligand-binding domain or ectodomain of Receptor activatorof nuclear factor-κB (RANK). In another aspect, the molecule includes aRANKL-binding amino acid sequence corresponding to SEQ ID NO: 95, 96,97, or 98. In an additional aspect, the immunomodulatory moiety inhibitsthe activity or function of Receptor activator of nuclear factor-κBligand (RANKL).

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, vascular endothelial growth factor (VEGF), cytotoxic T-lymphocyteantigen-4 (CTLA-4), CD25 (IL-2α receptor; IL-2αR), or CD4. In anadditional aspect, the immunomodulatory moiety includes an extracellularligand-binding domain or ectodomain of Receptor activator of nuclearfactor-κB (RANK). In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35,or 36.

The present invention provides novel targeted immunosuppressiveantibodies and fusion proteins that induce or promote immune toleranceby at least one of the following:

(i) inhibiting the activation of dendritic cells, T cells, and/or Bcells; and

(ii) promoting the development and/or suppressor function of regulatoryT cells and immunosuppressive myeloid DCs. These targetedimmunosuppressive molecules of the invention are designed to suppressunwanted or excessive immune or inflammatory responses in order to treatautoimmune or inflammatory diseases or prevent the rejection of atransplanted cell, tissue, or organ.

Targeted immunosuppressive antibodies and/or fusion proteins: Theaberrant activation of self-reactive T cells and/or breakdown of themechanisms of immune tolerance promotes the development of autoimmunitythat results in various diseases including type I diabetes mellitus,multiple sclerosis, systemic lupus erythematosus, inflammatory boweldisease, and rheumatoid arthritis. The targeted immunosuppressiveantibodies and/or fusion proteins of the invention are designed tosuppress unwanted or excessive immune or inflammatory responses andrestore or promote immune tolerance. Accordingly, the compositions andmethods of the invention have broad clinical relevance for the treatmentof diverse autoimmune or inflammatory diseases and preventing therejection of a transplanted cell, tissue, or organ grafts.

The targeted immunosuppressive antibodies and/or fusion proteins of theinvention provides their ability to inhibit the activity of targetedpro-inflammatory cytokines or immune cells while simultaneouslypromoting immune tolerance via the targeted delivery ofimmunosuppressive molecules that facilitate the development and/orfunction of regulatory T cells. These molecules of the present inventionare distinguished from and superior to existing therapeutic molecules inat least one of the following aspects: (i) The molecules of theinvention enable targeted delivery of immunosuppressive molecules toimmune cells or pro-inflammatory molecules in the milieu of the affectedcell, tissue or organ; (ii) The molecules of the invention can couplethe inhibition of the targeted pro-inflammatory molecule or immune cellwith the simultaneous delivery of an immunosuppressive molecule thatpromotes immune tolerance, thereby improving the suppression of immuneeffector cells; and (iii) The molecules of the invention can provide amechanism of simultaneously engaging two independent or synergisticmechanisms of immune tolerance or immune suppression.

Further, the targeted immunosuppressive antibodies and/or fusionproteins of the invention are distinguished from and superior toexisting therapeutic molecules in at least one of the following aspects:(i) To suppress unwanted or excessive immune or inflammatory responsesin order to treat autoimmune or inflammatory diseases; and (ii) Toprevent the rejection of a transplanted cell, tissue, or organ grafts.

In one aspect, the immunomodulatory moiety includes a sequence fromProgrammed death-1 ligand 1 (PD-L1) or Programmed death-1 ligand 2(PD-L2). In another aspect, the molecule includes a PD-1-binding aminoacid sequence corresponding to SEQ ID NO: 99, 100, or 101. In anadditional aspect, the immunomodulatory moiety increases the function ofPD-1.

In one aspect, the targeting moiety specifically binds to Tumor NecrosisFactor-a (TNF-α), and the immunomodulatory moiety includes a sequencefrom Programmed death-1 ligand 1 (PD-L1) or Programmed death-1 ligand 2(PD-L2). In an additional aspect, the targeting moiety includes anantibody that binds TNF-α, and the immunomodulatory moiety includes asequence from PD-1 ligand 1 (PD-L1 or B7-H1). In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 37.In another aspect, the targeting moiety includes an extracellularligand-binding domain of tumor necrosis factor receptor 2 (TNFR2), andthe immunomodulatory moiety includes a sequence from PD-1 ligand 1(PD-L1 or B7-H1). In another aspect, the molecule includes TNFR2Extracellular ligand binding domain, immunoglobulin Fc region, and asequence from PD-L1. In another aspect, the molecule includes an aminoacid sequence corresponding to SEQ ID NO: 38 or 39.

In one aspect, the targeting moiety includes an antibody or antibodyfragment that specifically binds to CD20, CD25, or CD4, and theimmunomodulatory moiety includes a sequence from Programmed death-1ligand 1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2). In anotheraspect, the molecule includes an amino acid sequence corresponding toSEQ ID NO: 40, 41, 42, 43, 44, or 45.

In one aspect, the targeting moiety includes the extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes a sequence from Programmed death-1 ligand 1 (PD-L1) orProgrammed death-1 ligand 2 (PD-L2). In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 46 or 47.

In one aspect, the targeting moiety includes transforming growthfactor-β (TGF-1) and immunoglobulin Fc region (IgG Cγ1), and theimmunomodulatory moiety includes a sequence from Programmed death-1ligand 1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2). In anadditional aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 48 or 49.

In one aspect, the immunomodulatory moiety includes a sequence fromtransforming growth factor-β (TGF-β). In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 99, 100, or101. In an additional aspect, the immunomodulatory moiety activates thesignaling function of transforming growth factor-β (TGF-β) receptor.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Tumor NecrosisFactor-α (TNF-α), and the immunomodulatory moiety includes a sequencefrom transforming growth factor-β (TGF-β). In an additional aspect, thetargeting moiety includes an antibody that binds TNF-α, and theimmunomodulatory moiety includes a sequence from TGF-β. In anotheraspect, the molecule includes an amino acid sequence corresponding toSEQ ID NO: 50. In one aspect, the targeting moiety includes anextracellular ligand-binding domain of tumor necrosis factor receptor 2(TNFR2). In another aspect, the molecule includes TNFR2 Extracellularligand binding domain, immunoglobulin Fc region, and a sequence fromtransforming growth factor-β (TGF-β). In another aspect, the moleculeincludes an amino acid sequence corresponding to SEQ ID NO: 51 or 52.

In one aspect, the targeting moiety includes an antibody or antibodyfragment that specifically binds to CD20, CD25 (IL-2α receptor; IL-2αR),or CD4, and the immunomodulatory moiety includes a sequence fromtransforming growth factor-β (TGF-β). In an additional aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 53,54, 55, 56, 57 or 58.

In one aspect, the targeting moiety includes an extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes a sequence from transforming growth factor-β (TGF-β). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 59 or 60.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to Tumor NecrosisFactor-α (TNF-α), and the immunomodulatory moiety includes anextracellular RANKL-binding domain or ectodomain of Receptor activatorof nuclear factor-α (RANK) or Osteoprotegerin (OPG). In an additionalaspect, the targeting moiety includes an antibody that binds TNF-α, andthe immunomodulatory moiety includes a sequence from an extracellularRANKL-binding domain or ectodomain of RANK. In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 61.In one aspect, the targeting moiety includes an extracellularligand-binding domain of tumor necrosis factor receptor 2 (TNFR2). Inanother aspect, the molecule includes TNFR2 Extracellular ligand bindingdomain, immunoglobulin Fc region, and a sequence from an extracellularRANK-binding domain or ectodomain of RANK. In another aspect, themolecule includes an amino acid sequence corresponding to SEQ ID NO: 62or 63.

In one aspect, the targeting moiety includes an extracellular domain ofCTLA-4 and immunoglobulin Fc region (IgG Cγ1), and the immunomodulatorymoiety includes an extracellular RANKL-binding domain or ectodomain ofReceptor activator of nuclear factor-κB (RANK) or Osteoprotegerin (OPG).In another aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 64 or 65.

In one aspect, the targeting moiety includes includes a sequence fromtransforming growth factor-β (TGF-β) and immunoglobulin Fc region (IgGCγ1), and the immunomodulatory moiety includes an extracellularRANKL-binding domain or ectodomain of Receptor activator of nuclearfactor-κB (RANK). In another aspect, the molecule includes an amino acidsequence corresponding to SEQ ID NO: 66 or 67.

In one aspect, the targeting moiety includes a sequence from Programmeddeath-1 ligand 1 (PD-L1) and immunoglobulin Fc region (IgG Cγ1), and theimmunomodulatory moiety includes an extracellular RANKL-binding domainor ectodomain of Receptor activator of nuclear factor-κB (RANK). Inanother aspect, the molecule includes an amino acid sequencecorresponding to SEQ ID NO: 68 or 69.

In various aspects, the molecule is fused or directly linked to one ormore antigen, antigenic determinant, or epitope.

In another embodiment, the present invention provides a compositionincluding the molecule of the invention and a cell, wherein the cell isa tumor cell, immune cell, or dendritic cell.

In another embodiment, the present invention provides a method ofcounteracting or overcoming immune tolerance. The method includesadministering to a subject in need thereof one or more molecule of theinvention.

In another embodiment, the present invention provides a method ofpreventing or treating a neoplastic disease. In one aspect, theneoplastic disease is a non-T cell malignancy which does not express CD4on the tumor cell. In one embodiment, the method includes administrationto a subject in need thereof an antibody that targets and depletes CD4+regulatory T cells (Tregs) in combination with a cytotoxic anticancertherapy. In one aspect, the antibody that targets and depletes Tregs isan anti-CD4 antibody. In various aspects, the cytotoxic anticancertherapy includes a chemotherapeutic molecule, tumor-targeted antibody,small molecule kinase inhibitor, hormonal agent, or tumor-targetedcytotoxic agent, anti-angiogenic agent or any combination thereof. Inanother aspect, the cytotoxic anticancer therapy includes ionizingradiation, ultraviolet radiation, cryoablation, thermal ablation, orradiofrequency ablation.

In another embodiment, the method includes administration to a subjectin need thereof an antibody or molecule that targets and depletes CD4+regulatory T cells (Tregs) in combination with an immunostimulatoryantibody, fusion protein, peptide or ligand that targets CTLA-4, PD1,PD-1L, RANKL, TGF-β, GITR, 4-1BB, OX-40, or Toll-like receptors (TLR1-10). In one aspect, the TLR-agonist comprises an activator of TLR-8 orTLR-9. In one aspect, the TLR agonist comprises an immunostimulatorynucleic acid sequence containing CpG nucleotides. In one aspect, theantibody that targets and depletes Tregs is an anti-CD4 antibody.

In another embodiment, the present invention provides a method ofpreventing or treating a neoplastic disease. The method includesadministration to a subject in need thereof one or more molecule of theinvention. In various aspects, the subject is administered one or moremolecule of the invention in combination with another anticancertherapy. In one aspect, the anticancer therapy includes achemotherapeutic molecule, antibody, small molecule kinase inhibitor,hormonal agent, cytotoxic agent, targeted therapeutic agent, oranti-angiogenic agent. In another aspect, the anticancer therapyincludes ionizing radiation, ultraviolet radiation, cryoablation,thermal ablation, or radiofrequency ablation. In another aspect, thesubject is administered one or more molecule of the invention incombination with an antibody or molecule that inhibits the production orfunction of regulatory T cells (Tregs) or depletes the number of Tregs.In one aspect, the antibody that targets and depletes Tregs is ananti-CD4 antibody. In another aspect, the molecule that counteracts thefunction of Tregs is an antibody, fusion protein, peptide or ligand thattargets CTLA-4, PD1, PD-1L, RANKL, TGF-β, GITR. In another aspect, themolecule that counteracts the function of Tregs is an antibody or fusionprotein or ligand that targets 4-1BB or OX-40. In another aspect, themolecule that counteracts the function of Tregs is an agonist ofToll-like receptors (TLR 1-10). In one aspect, the TLR-agonist comprisesan activator of TLR-8 or TLR-9. In one aspect, the TLR agonist is animmunostimulatory nucleic acid sequence containing CpG nucleotides.

In one aspect the chemotherapeutic agent is a topoisomerase-interactingagent, anthracycline, doxorubicin, mitoxantrone, camptothecin,camptothecin analogue, irinotecan, epipodophyilotoxin, etoposide,alkylating agent, cyclophosphamide, cisplatin, cisplatin analogue,oxaliplatin, antimetabolite, fluoropyrimidine analogue, 5-fluorouracil,gemcitabine, azacytidine, antimicrotubule agent, taxane, paclitaxel, ordocetaxel.

In another embodiment, the subject is administered one or more moleculeof the invention in combination with any vaccine. In another aspect, thevaccine includes a tumor antigen, tumor-associated antigen, tumorepitope, tumor antigen-containing fusion protein, tumor cell, ordendritic cell. In another aspect, the vaccine includes a pathogenantigen, pathogen-associated antigen, pathogen epitope, or pathogenantigen-containing fusion protein. In one aspect, the vaccine includes asurrogate CD4+ T cell helper epitope from tetanus toxin. In one aspect,the CD4+T helper sequence contains a domain of tetanus toxin fragment C(pDOM1). In one aspect, the pDOM sequence is fused to acell-permeabilizing cationic polypeptide (e.g. Arginine-9). In anotheraspect, the Arg9-pDOM sequence is fused to a specific antigen comprisingthe vaccine.

In another embodiment, the present invention provides a method fortreating immune cells wherein the cells are contacted ex vivo or invitro with a molecule of the invention. In another embodiment, thepresent invention provides a method of treatment of a neoplasticdisease. The method includes administering to a subject in need thereofa composition of immune cells contacted with a molecule of theinvention.

In another embodiment, the present invention provides a method ofinducing or promoting immune tolerance. The method includesadministering to a subject in need thereof one or more molecule of theinvention.

In another embodiment, the present invention provides a method ofpreventing or treating an autoimmune or inflammatory disease includingadministering to a subject in need thereof one or more molecule of theinvention. In one aspect, the subject is administered one or moremolecule of the invention in combination with another anti-inflammatoryor immunosuppressive therapy. In another embodiment, the presentinvention provides a method of treatment of immune cells wherein thecells are contacted ex vivo or in vitro with a molecule of theinvention. In another embodiment, the present invention provides amethod of treating an autoimmune or inflammatory disease or preventingrejection of grafted cells or tissue. The method includes administeringto a subject in need thereof a composition of immune cells contactedwith a molecule of the invention.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

As used herein “immune cells” or “immune effector cells” include Tlymphocytes, B lymphocytes, natural killer (NK) cells, NKT cells,monocytes, macrophages, dendritic cells (DC), antigen presenting cells(APC).

As used herein, “neoplasm” or “tumor”, including grammatical variationsthereof, means new and abnormal growth of tissue, which may be benign orcancerous. In a related aspect, the neoplasm is indicative of aneoplastic disease or disorder, including but not limited, to variouscancers. For example, such cancers can include prostate, pancreatic,biliary, colon, rectal, liver, kidney, lung, testicular, breast,ovarian, pancreatic, brain, and head and neck cancers, melanoma,sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

A used herein, “subject,” including grammatical variations thereof,means a human or vertebrate animal including a dog, cat, horse, cow,pig, sheep, goat, chicken, monkey, rat, and mouse.

As used herein, “targeting moiety” refers to a molecule that has theability to localize and bind to a specific molecule or cellularcomponent. The targeting moiety can be an antibody, antibody fragment,scFv, Fc-containing polypeptide, fusion antibody, polypeptide, peptide,aptamer, ligand, nucleic acid, or any combination thereof. In oneembodiment, a targeting moiety can bind to a molecule present in a cellor tissue. In one aspect, the targeting moiety can bind a molecule in adiseased cell or tissue, such as a cancer cell or tumor. In, anotheraspect, the targeting molecule can bind a normal cell or tissue, such asan immune cell. In another aspect, the targeting moiety can bind acellular or extracellular molecule that modulates the immune response.In another aspect, the targeting moiety binds a growth factor receptor,growth factor, cytokine receptor, cytokine, or cell surface molecule.

In another embodiment, the targeting moiety is a tumor-targeting moiety.The tumor-targeting moiety can bind a component of a tumor cell or bindin the vicinity of a tumor cell (e.g., tumor vasculature or tumormicroenvironment). In one embodiment, the tumor targeting moiety bindsto a component of a tumor cell, tumor microenvironment, tumorvasculature, tumor-associated lymphocyte, tumor antigen,tumor-associated antigen, tumor cell surface molecule, tumor antigenicdeterminant, tumor antigen-containing fusion protein, tumor-associatedcell, tumor-associated immune cell, or tumor vaccine.

For example, in various embodiments, a targeting moiety is specific foror binds to a molecule or component, which includes but is not limitedto, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1), ErbB-2(HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-likegrowth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs),IGFR ligand family (IGF-1R); platelet derived growth factor receptor(PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor(FGFR) family, FGFR ligand family, vascular endothelial growth factorreceptor (VEGFR) family, VEGF family; HGF receptor family: TRK receptorfamily; ephrin (EPH) receptor family; AXL receptor family; leukocytetyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptorfamily; discoidin domain receptor (DDR) family; RET receptor family; KLGreceptor family; RYK receptor family; MuSK receptor family; Transforminggrowth factor alpha (TGF-α), TGF-α receptor; Transforming growthfactor-beta (TGF-β), TGF-β receptor; Interleukin 13 receptor alpha2chain (IL13Ralpha2), Interleukin-6 (IL-6), 1L-6 receptor, Interleukin-4,IL-4 receptor, Cytokine receptors, Class I (hematopoietin family) andClass II (interferon/IL-10 family) receptors, tumor necrosis factor(TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily(TNTRSF), death receptor family, TRAIL-receptor; cancer-testis (CT)antigens, lineage-specific antigens, differentiation antigens,alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl)fusion products, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8),beta-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependentkinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2,Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloid leukemia 1gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, lowdensity lipid receptor/GDP-L fucose: beta-Dgalactose2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein, HLA-A2, arginineto isoleucine exchange at residue 170 of the alpha-helix of thealpha2-domain in the HLA-A2 gene (HLA-A*201-R170I), MLA-A11, heat shockprotein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitousmutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP),neo-PAP, Myosin class 1, NFYC, OGT, OS-9, pml-RARalpha fusion protein,PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1,SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE,BAGE-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetylgiucosaminyl transferase V, MGATS), HERV-K-MEL, KK-LC, KM-HN-1, LAGE,LAGE-1, CTL-recognixed antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1),MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1,MAGE-C2, mucin 1 (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117(S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2,SAGE, Sp17, SSX-1,2,3,4, TRP2-1NT2, carcino-embryonic antigen (CEA),Kallikfein 4, mammaglobm-A, OA1, prostate specific antigen (PSA),prostate specific membrane antigen, TRP-1/gp75, TRP-2, adipophilin,interferon inducible protein absent in nielanorna 2 (AIM-2), BING-4,CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3,fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250intestinalcarboxyl esterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUCI,p53 (TP53), PBF, FRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1,survivin (BIRCS), human telomerase reverse transcriptase (hTERT),telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2,PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14,HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1,TEX 15, FATE, TPTE, immunoglobulin idiotypes, Bence-Jones protein,estrogen receptors (ER), androgen receptors (AR), CD40, CD30, CD20,CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancerantigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen125 (CA 125), cancer antigen 19-9 (CA 19-9), beta-human chorionicgonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen,neuron-specific enoJase, heat shock protein gp96, GM2, sargramostim,CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognizedby T cells 4 (ART-4), carcinoembryogenic antigen peptide-1 (CAP-1),calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B),human signet ring tumor-2 (HST-2), Human papilloma virus (HPV) proteins(HPV-E6, HPV-E7, major or minor capsid antigens, others), Epstein-Barrvims (EBV) proteins (EBV latent membrane proteins—LMP1, LMP2; others),Hepatitis B or C virus proteins, and HIV proteins. A composition of theinvention can further include the foregoing as a peptide/polypeptideand/or encoding the same.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, or Fc-containing polypeptide that specifically binds acomponent of a tumor cell, tumor antigen, tumor vasculature, tumormicroenvironment, or tumor-infiltrating immune cell. In one aspect, thetargeting moiety specifically binds epidermal growth factor receptor(EGFR1, Erb-B1), HER2/neu (Erb-B2), CD20, Vascular endothelial growthfactor (VEGF), insulin-like growth factor receptor (IGF-1R),TRAIL-receptor, epithelial cell adhesion molecule, carcino-embryonicantigen, Prostate-specific membrane antigen, Mucin-1, CD30, CD33, CD40,or a combination thereof.

Examples of antibodies which can be incorporated into compositions andmethods disclosed herein include, but are not limited, to antibodiessuch as trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2mAb); cetuximab (chimeric monoclonal antibody to epidermal growth factorreceptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFRantibody); Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb);MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanizedanti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptorbispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb);Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb);Tositumumab-I131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb);Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab(anti-VEGF mAb); Aflibercept (extracellular domains of VEGFR1 and VEGFR2fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fusedto IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin(anti-CD33 mAb); Alemtuzumab (anti-Campath-I/CD52 mAb); Brentuximabvedotin (anti-CD30 mAb); Catumaxomab (bispecific mAb that targetsepithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb);Girentuximab (anti-Carbonic anhydrase ix); or Farletuzumab (anti-folatereceptor). Other examples include antibodies such as Panorex™ (17-1A)(murine monoclonal antibody); Panorex (@ (17-1A) (chimeric murinemonoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope)(with BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab,humanized 13′ 1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotypic mousemAb); 3622W94 mAb that binds to EGP40 (17-1A) pancarcinoma antigen onadenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART M195 Ab,humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab); TNT(chimeric mAb to histone antigens); TNT (chimeric mAb to histoneantigens); Gliomab-H (Monoclonals Humanized Abs); GNI-250 Mab; EMD-72000(chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); andMDX-260 bispecific, targets GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364Ab or ImmuRAIT-CEA. Examples of antibodies include those disclosed inU.S. Pat. Nos. 5,736,167, 7,060,808, and 5,821,337.

In one embodiment, the targeting moiety specifically binds a componentof a regulatory T cell, myeloid suppressor cell, or dendritic cell. Inanother aspect, the targeting moiety specifically binds one of thefollowing molecules: CD4; CD25 (IL-2α receptor; IL-2αR); cytotoxicT-lymphocyte antigen-4 (CTLA-4; CD152); Interleukin-10 (IL-10);Transforming growth factor-beta receptor (TGF-βR); Transforming growthfactor-beta (TGF-β); Programmed Death-1 (PD-1); Programmed death-1ligand (PD-L1 or PD-L2); Receptor activator of nuclear factor-α (RANK);Receptor activator of nuclear factor-κB (RANK) ligand (RANKL); LAG-3;glucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; TNFRSF18); or Interleukin-4 receptor (IL-4R). In one aspect,the targeting moiety is an agonist that increases the function of thetargeted molecule. In another aspect, the targeting moiety is anantagonist that inhibits the function of the targeted molecule.

In one aspect, the targeting moiety binds a specific cytokine, cytokinereceptor, co-stimulatory molecule, co-inhibitory molecule, orimmunomodulatory receptor that modulates the immune system. In anotheraspect, the targeting moiety specifically binds one of the followingmolecules: tumor necrosis factor (TNF) superfamily; tumor necrosisfactor-α (TNF-α); tumor necrosis factor receptor (TNFR) superfamily;Interleukin-12 (IL-12); IL-12 receptor; 4-1BB (CD137); 4-1BB ligand(4-1BBL; CD137L); OX40 (CD134; TNR4); OX40 ligand (OX40L; CD40; CD40ligand (CD40L); CTLA-4; Programmed death-1 (PD-1); PD-1 ligand 1 (PD-L1;B7-H1); or PD-1 ligand 2 (PD-L2; B7-DC); B7 family; B7-1 (CD80); B7-2(CD86); B7-H3; B7-H4; GITR/AITR; GITRL/AITRL; BTLA; CD70; CD27; LIGHT;HVEM; Toll-like receptor (TLR)(TLR 1,2,3,4,5,6,7,8,9, 10). In oneaspect, the targeting moiety is an agonist that increases the functionof the targeted molecule. In another aspect, the targeting moiety is anantagonist that inhibits the function of the targeted molecule.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, Fc-containing polypeptide, or peptide that specificallybinds a component of a regulatory T cell, myeloid suppressor cell, ordendritic cell. In another aspect, the targeting moiety includes anantibody, antibody fragment, scFv, or Fc-containing polypeptide thatspecifically binds a cytokine, cytokine receptor, co-stimulatorymolecule, or co-inhibitory molecule that modulates the immune system. Inanother aspect, the targeting moiety specifically binds one of thefollowing molecules: CD4; CD25 (IL-2α receptor; IL-2αR); cytotoxicT-lymphocyte antigen-4 (CTLA-4; CD152); Interleukin-10 (IL-10);Transforming growth factor-beta receptor (TGF-βR); Transforming growthfactor-beta (TGF-β); Programmed Death-1 (PD-1); PD-1 ligand 1 (PD-L1;B7-H1); PD-1 ligand 2 (PD-L2; B7-DC); Receptor activator of nuclearfactor-κB (RANK); Receptor activator of nuclear factor-κB (RANK) ligand(RANKL); LAG-3; glucocorticoid-induced tumor necrosis factor receptorfamily-related gene (GITR; TNFRSF18); Interleukin-4 receptor (IL-4R);tumor necrosis factor (TNF) superfamily; tumor necrosis factor-α(TNF-α); tumor necrosis factor receptor (TNFR) superfamily;Interleukin-12 (IL-12); IL-12 receptor; 4-1BB (CD137); 4-1BB ligand(4-1BBL; CD137L); OX40 (CD134; TNR4); OX40 ligand (OX40L; CD40; CD40ligand (CD40L); CTLA-4; B7 family; B7-1 (CD80); B7-2 (CD86); B7-H3;B7-H4; GITR/AI GITRL/AITRL; BTLA; CD70; CD27; LIGHT; or HVEM. In oneaspect, the targeting moiety is an agonist that increases the functionof the targeted molecule. In another aspect, the targeting moiety is anantagonist that inhibits the function of the targeted molecule.

Examples of antibodies which can be incorporated into compositions andmethods disclosed herein include, but are not limited, to antibodiessuch as Zanulimumab (anti-CD4 mAb), Keliximab (anti-CD4 mAb); Ipilimumab(MDX-101; anti-CTLA-4 mAb); Tremilimumab (anti-CTLA-4 mAb); (Daclizumab(anti-CD25/IL-2R mAb); Basiliximab (anti-CD25/IL-2R mAb); MDX-1106(anti-PD1 mAb); antibody to GITR; GC1008 (anti-TGF-β antibody);metelimumab/CAT-192 (anti-TGF-β antibody); lerdelimumab/CAT-152(anti-TGF-β antibody); ID11 (anti-TGF-β antibody); Denosumab (anti-RANKLmAb); BMS-663513 (humanized anti-4-1BB mAb); SGN-40 (humanized anti-CD40mAb); CP870,893 (human anti-CD40 mAb); Infliximab (chimeric anti-TNFmAb; Adalimumab (human anti-TNF mAb); Certolizumab (humanized Fabanti-TNF); Golimumab (anti-TNF); Etanercept (Extracellular domain ofTNFR fused to IgG1 Fc); Belatacept (Extracellular domain of CTLA-4 fusedto Fc); Abatacept (Extracellular domain of CTLA-4 fused to Fc);Belimumab (anti-B Lymphocyte stimulator); Muromonab-CD3 (anti-CD3 mAb);Otelixizumab (anti-CD3 mAb); Teplizumab (anti-CD3 mAb); Tocilizumab(anti-IL6R mAb); REGN88 (anti-IL6R mAb); Ustekinumab (anti-IL-12/23mAb); Briakinumab (anti-IL-12/23 mAb); Natalizumab (anti-a4 integrin);Vedolizumab (anti-α4 β7 integrin mAb); T1 h (anti-CD6 mAb); Epratuzumab(anti-CD22 mAb); Efalizumab (anti-CD11a mAb); and Atacicept(extracellular domain of transmembrane activator and calcium-modulatingligand interactor fused with Fc).

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an “immunomodulatory moiety”. As usedherein, “immunomodulatory moiety” refers to a ligand, peptide,polypeptide, or Fc-containing polypeptide that binds a specificcomponent of a regulatory T cell, myeloid suppressor cell, or dendriticcell and modulates the number or function of Tregs or myeloid suppressorcells. In an additional aspect, the “immunomodulatory moiety”specifically binds a cytokine, cytokine receptor, co-stimulatorymolecule, or co-inhibitory molecule that modulates the immune system. Inanother aspect, the immunomodulatory moiety specifically binds one ofthe following molecules: Transforming growth factor-beta receptor(TGF-βR); Transforming growth factor-beta (TGF-β); Programmed Death-1(PD-1); PD-1 ligand 1 (PD-L1; B7-H1); PD-1 ligand 2 (PD-L2; B7-DC);Receptor activator of nuclear factor-κB (RANK); or Receptor activator ofnuclear factor-κB (RANK) ligand (RANKL); or vascular endothelial growthfactor (VEGF). In another aspect, the immunomodulatory moietyspecifically binds one of the following molecules:glucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; AITR; TNFRSF18); GITRL/AITRL; 4-1BB (CD137); 4-1BB ligand(4-1BBL; CD137L); OX40 (CD134; TNR4); OX40 ligand (OX40L); B7-H3; B7-H4;BTLA; CD40; CD40 ligand (CD40L); CD70; CD27; LIGHT; or HVEM. In anotheraspect, the immunomodulatory moiety specifically binds one of thefollowing molecules: tumor necrosis factor-α (TNF-α); Interleukin-12(IL-12); IL-12R; Interleukin-10 (IL-10); IL-10R. In another aspect, theimmunomodulatory moiety comprises an extracellular domain of CTLA-4. Inone aspect, the immunomodulatory moiety is an agonist that increases thefunction of the bound molecule. In another aspect, the immunomodulatorymoiety is an antagonist that inhibits the function of the targetedmolecule.

In another aspect, the immunomodulatory moiety comprises anextracellular domain or ligand-binding sequence of one of the followingreceptors: Transforming growth factor-beta receptor (TGF-βRII,TGF-βRIIb, or TGF-βRIII); Programmed Death-1 (PD-1); Receptor activatorof nuclear factor-κB (RANK); vascular endothelial growth factor receptor(VEGFR1 or VEGFR2); or IL-10R. In another aspect, the immunomodulatorymoiety comprises an extracellular domain or ligand-binding sequence ofone of the following receptors: tumor necrosis factor receptor 2(TNFR2); 4-1BB (CD137); OX40 (CD134; TNR4); CD40; IL-12R; orglucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; AITR; TNFRSF18). In an additional aspect, the extracellulardomain of the specific receptor binds the cognate ligand and inhibitsthe interaction of the ligand with its native receptor.

In another aspect, the immunomodulatory moiety comprises one or more ofthe following ligands or active ligand fragments: Transforming growthfactor-beta (TGF-β); PD-1 ligand 1 (PD-L1); PD-1 ligand 2 (PD-L2); orIL-10. In another aspect, the immunomodulatory moiety comprises one ormore of the following ligands or active ligand fragments: 4-1BB ligand(4-1BBL; CD137L); OX40 ligand (OX40L); IL-12; CD40L; or GITRL/AITRL.

In another aspect, the immunomodulatory moiety is fused to theC-terminus of the targeting moiety. In another aspect, theimmunomodulatory moiety is fused to the N-terminus of the targetingmoiety. In one aspect, the fusion molecule is represented by X-Fc-Y,wherein X is the targeting moiety, Fc is an immunoglobulin Fc region,and Y is the immunomodulatory moiety. In another aspect, the fusionmolecule is represented by Y-Fc-X, wherein X is the targeting moiety,and Y is the immunomodulatory moiety. In one aspect, the targetingmoiety may additionally be an immunomodulatory moiety.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, or Fc-containing polypeptide that specifically binds acomponent of a tumor cell, tumor antigen, tumor vasculature, tumormicroenvironment, or tumor-infiltrating immune cell, and theimmunomodulatory moiety comprises an extracellular domain orligand-binding sequence of one of the following receptors: Transforminggrowth factor-beta receptor (TGF-βRII, TGF-βRIIb, or TGF-βRIII);Programmed Death-1 (PD-1); Receptor activator of nuclear factor-κB(RANK); vascular endothelial growth factor receptor (VEGFR1 or VEGFR2);or IL-10R.

In one aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, or Fc-containing polypeptide that specifically binds acomponent of a tumor cell, tumor antigen, tumor vasculature, tumormicroenvironment, or tumor-infiltrating immune cell, and theimmunomodulatory moiety comprises one or more of the following ligandsor active ligand fragments: 4-1BB ligand (4-1BBL; CD137L); OX40 ligand(OX40L); IL-12; CD40L; or GITRL/AITRL.

In another aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, Fc-containing polypeptide or ligand that binds aspecific component of a regulatory T cell, myeloid suppressor cell, ordendritic cell, and the immunomodulatory moiety comprises anextracellular domain or ligand-binding sequence of one of the followingreceptors: Transforming growth factor-beta receptor (TGF-βRII,TGF-βRIIb, or TGF-βRIII); Programmed Death-1 (PD-1); Receptor activatorof nuclear factor-κB (RANK); or IL-10R. In another aspect, theimmunomodulatory moiety comprises one or more of the following ligandsor active ligand fragments: 4-1BB ligand (4-1BBL; CD137L); OX40 ligand(OX40L); IL-12; CD40L; or GITRL/AITRL. In another aspect, the specifictargeted component of a regulatory T cell, myeloid suppressor cell, ordendritic cell is one of the following molecules: CD4; CD25 (IL-2αreceptor; IL-2αR); cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152);Interleukin-10 (IL-10); Transforming growth factor-beta (TGF-β);Programmed Death-1 (PD-1); Programmed death-1 ligand (PD-L1 or PD-L2);Receptor activator of nuclear factor-κB (RANK) ligand (RANKL); LAG-3;glucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; TNFRSF18); or Interleukin-4 receptor (IL-4R).

In another aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, Fc-containing polypeptide or ligand that binds one ofthe following: CTLA-4; 4-1BB (CD137); OX40 (CD134; TNR4); CD40; IL-12R;or glucocorticoid-induced tumor necrosis factor receptor family-relatedgene (GITR; AITR; TNFRSF18); and the immunomodulatory moiety comprises adifferent molecule selected from the following: (i) an extracellulardomain or ligand-binding sequence of one of the following receptors:Transforming growth factor-beta receptor (TGF-βRII, TGF-βRIIb, orTGF-βRIII); Programmed Death-1 (PD-1); Receptor activator of nuclearfactor-κB (RANK); or IL-10R; or (ii) Fc-containing polypeptide or ligandthat binds one of the following: CTLA-4; 4-1BB (CD137); OX40 (CD134;TNR4); CD40; IL-12R; or GITR (AITR; TNFRSF18).

In another aspect, the targeting moiety and immunomodulatory moiety aretwo different molecules selected from any of the following: an antibody,antibody fragment, scFv, Fc-containing polypeptide or ligand that bindsTGF-β, CTLA-4, PD-1, 4-1BB (CD137), OX40 (CD134; TNR4), CD40; IL-12R, orGITR/AITR (TNFRSF18), or Toll-like receptor (TLR); an extracellulardomain or ligand-binding sequence of Transforming growth factor-betareceptor (TGF-βRII, TGF-βRIIb, or TGF-βRIII), Programmed Death-1 (PD-1),Receptor activator of nuclear factor-κB (RANK), or IL-10R. In oneaspect, the fusion molecule is represented by X-Fc-Y, wherein X is animmunomodulatory targeting moiety and Y is a different immunomodulatorymoiety.

In another aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, Fc-containing polypeptide that binds one of thefollowing molecules: CD4; CD25 (IL-2α receptor; IL-2αR); or CD20; andthe immunomodulatory moiety comprises one of the following ligands oractive ligand fragments: Transforming growth factor-beta (TGF-β); PD-1ligand 1 (PD-L1); PD-1 ligand 2 (PD-L2); or IL-10.

In another aspect, the targeting moiety includes an antibody, antibodyfragment, scFv, Fc-containing polypeptide that binds tumor necrosisfactor-α (TNF-α), Interleukin-12 (IL-12), IL-6R, B-lymphocytestimulator, CD11a, CD6, or CD22; and the immunomodulatory moietycomprises one of the following: (i) ligands or active ligand fragmentsof Transforming growth factor-beta (TGF-β), PD-1 ligand 1 (PD-L1), orIL-10; or (ii) an extracellular domain or ligand-binding fragment ofRANK, 4-1BB (CD137), OX40 (CD134; TNR4), CD40, IL-12R or GITR/AITR(TNFRSF18).

In another aspect, the targeting moiety comprises the extracellulardomain of CTLA-4 fused to immunoglobulin Fc, and the immunomodulatorymoiety comprises one of the following: (i) ligands or active ligandfragments of Transforming growth factor-beta (TGF-β), PD-1 ligand 1(PD-L1), or IL-10; or (ii) ligand-binding fragment of TNFR2, RANK, 4-1BB(CD137), OX40 (CD134; TNR4), CD40, IL-12R or GITR/AITR (TNFRSF18).

In another aspect, the targeting moiety and immunomodulatory moiety aretwo different molecules selected from any of the following: an antibody,antibody fragment, scFv, Fc-containing polypeptide that binds tumornecrosis factor-α (TNF-α), Interleukin-12 (IL-12), IL-6R, B-lymphocytestimulator, CD11a, CD6, or CD22; a ligand-binding fragment of TNFR2,RANK, 4-1BB (CD137), OX40 (CD134; TNR4), CD40, IL-12R or GITR/AITR(TNFRSF18); ligands or active ligand fragments of Transforming growthfactor-beta (TGF-β), PD-1 ligand 1 (PD-L1), or IL-10; or CTLA-4-Fc. Inone aspect, the fusion molecule is represented by X-Fc-Y, wherein X isthe immunomodulatory targeting moiety and Y is a differentimmunomodulatory moiety.

Antibodies: In one embodiment, the targeting moiety or fusion protein isan immunoglobulin. As used herein, the term “immunoglobulin” includesnatural or artificial mono- or polyvalent antibodies including, but notlimited to, polyclonal, monoclonal, multispecific, human, humanized orchimeric antibodies, single chain antibodies, Fab fragments. F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin ion can be ofany type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulinmolecule.

An antibody as disclosed herein includes an antibody fragment, such as,but not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdfv) and fragmentsincluding either a VL or VH domain. In one embodiment, the targetingmoiety is an antibody or scFv.

An antigen-binding antibody fragment, including single-chain antibody,may include the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, CH1, CH2, and CH3domains. An antigen-binding fragment can also include any combination ofvariable region(s) with a hinge region, CHI, CH2, and CH3 domains. Alsoincludes is a Fc fragment, antigen-Fc fusion proteins, and Fc-targetingmoiety. The antibody may be from any animal origin including birds andmammals. In one aspect, the antibody is, or derived from, a human,murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken. Further, such antibody may be a humanizedversion of an antibody. The antibody may be monospecific, bispecific,trispecific, or of greater multispecificity.

The antibody herein specifically include a “chimeric” antibody in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984)Proc. Natl. Acad. Sci. USA. 81:6851-6855). A chimeric antibody ofinterest herein includes “primatized” antibodies including variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc) and human constant region sequences.

Various methods have been employed to produce antibodies. Hybridomatechnology, which refers to a cloned cell line that produces a singletype of antibody, uses the cells of various species, including mice(murine), hamsters, rats, and humans. Another method to prepare anantibody uses genetic engineering including recombinant DNA techniques.For example, antibodies made from these techniques include, amongothers, chimeric antibodies and humanized antibodies. A chimericantibody combines DNA encoding regions from more than one type ofspecies. For example, a chimeric antibody may derive the variable regionfrom a mouse and the constant region from a human. A humanized antibodycomes predominantly from a human, even though it contains nonhumanportions. Like a chimeric antibody, a humanized antibody may contain acompletely human constant region. But unlike a chimeric antibody, thevariable region may be partially derived from a human. The nonhuman,synthetic portions of a humanized antibody often come from CDRs inmurine antibodies. In any event, these regions are crucial to allow theantibody to recognize and bind to a specific antigen.

In one embodiment, a hybridoma can produce a targeted fusion proteincomprising a targeting moiety and an immunomodulatory moiety. In oneembodiment, a targeting moiety comprising an antibody, antibodyfragment, or polypeptide is linked or fused to an immunomodulatorymoiety consisting of a polypeptide, with a linker or without a linker.The linker can be an amino acid linker. In one embodiment, a linker is(GGGGS)n (SEQ ID NO: 123) wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. Forexample, GGGGSGGGGSGGGGS (SEQ ID NO: 104). In another embodiment, alinker is EPKSCDK (SEQ ID NO: 105). In another embodiment, a linker isIEGRDMD (SEQ. ID. NO: 106). In various aspects, the length of the linkermay be modified to optimize binding of the target moiety or the functionof the immunomodulatory moiety. In various aspects, the immunomodulatorymoiety is a polypeptide that is fused to the C-terminus of the Fc regionof the heavy chain of a targeting antibody or Fc-containing fusionprotein. In another aspect, the immunomodulatory moiety is a polypeptidethat is fused to the C-terminus of the light chain of a targetingantibody. In another aspect, the fusion protein comprises an X-Fc-Ysequence, wherein X is a targeting polypeptide and Y is animmunomodulatory polypeptide.

For example, a hybridoma can produce the polypeptides corresponding toSEQ. ID. NO: 1-69.

An antibody fragment can include a portion of an intact, antibody, e.g.including the antigen-binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; Fcfragments or Fc-fusion products; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragment(s).

An intact antibody is one which includes an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof tor any other modified Fc (e.g.glycosylation or other engineered Fc).

The intact antibody may have one or more “effector functions” whichrefer to those biological activities attributable to the Fc region (anative sequence Fc region or amino acid sequence variant Fc region orany other modified Fc region) of an antibody. Examples of antibodyeffector functions include Clq binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g., B cell receptor (BCR); and cross-presentation ofantigens by antigen presenting cells or dendritic cells. In oneembodiment, the targeting antibody or Fc-containing fusion proteinfacilitates focused or preferential delivery of a immunomodulatorymoiety to a target cell. In another aspect, a targeting antibody caninduce death of the targeted cell or sensitize it to immunecell-mediated cytotoxicity. In another aspect, the Fc-fusion protein orantibody can facilitate delivery of the immunomodulatory moiety orimmunogenic apoptotic material from antibody-bound tumor targets, orboth, to an antigen presenting cells (APC) via interactions betweentheir Fc and Fc receptors (on APC).

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgC3, IgG4, IgA, and IgA2. The heavy-chainconstant domains (hat correspond to the different classes of antibodiesare called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ)respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

Peptides: In some aspects of the invention the targeting moiety orimmunomodulatory moiety is a peptide or polypeptide. A peptide includesany analog, fragment or chemical derivative of a peptide whose aminoacid residue sequence is shown herein. Therefore, a present peptide canbe subject to various changes, substitutions, insertions, and deletionswhere such changes provide for certain advantages in its use. In thisregard, a peptide of this invention corresponds to, rather than isidentical to, the sequence of a recited peptide where one or morechanges are made and it retains the ability to function as theunmodified peptide in one or more of the assays.

The term “analog” includes any peptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the activity asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is disclosed herein.

As used herein “a tumor targeting peptide” includes polymers containingfewer than 100 amino acids, where the polymer specifically binds to acellular component of a tumor cell, tumor vasculature, and/or acomponent of a tumor microenvironment.

A peptide of the present invention can be synthesized by any of thetechniques that are known to those skilled in “the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. An excellent summary of themany techniques available can be found in Steward et al., “Solid PhasePeptide Synthesis”* W.H. Freeman Co., San Francisco, 1969; Bodanszky, etal., “Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”. Vol. 2. p. 46, AcademicPress (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969;Fields et al. Int. J. Peptide Protein Res., 35:161-214, 1990; and U.S.Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder etal., “The Peptides”, Vol. 1, Academic Press (New York), 1965 forclassical solution synthesis. Appropriate protective groups usable insuch synthesis are described in the above texts and in J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, New York, 1973.

Aptamers: In one aspect of the invention, the targeting moiety is anaptamer. In various embodiments, an aptamer is specific for a moleculeon a tumor cell, tumor vasculature, and/or a tumor microenvironment. Theterm “aptamer” includes DNA, RNA or peptides that are selected based onspecific binding properties to a particular molecule. For example, anaptamer(s) can be selected for binding a particular gene product in atumor cell, tumor vasculature, tumor microenvironment, and/or an immunecell, as disclosed herein, where selection is made by methods known inthe art and familiar to one of skill in the art. Subsequently, saidaptamer(s) can be administered to a subject to modulate or regulate animmune response.

Some aptamers having affinity to a specific protein, DNA, amino acid andnucleotides have been described (e.g., K. Y. Wang, et al., Biochemistry32:1899-1904 (1993); Pitner et al., U.S. Pat. No. 5,691,145: Gold, etal., Ann. Rev. Biochem. 64:763-797 (1995); Szostak et al., U.S. Pat. No.5,631,146). High affinity and high specificity binding aptamers havebeen derived from combinatorial libraries (supra, Gold, et al.).Aptamers may have high affinities, with equilibrium dissociationconstants ranging from micromolar to sub-nanomolar depending on theselection used, aptamers may also exhibit high selectivity, for example,showing a thousand fold, discrimination between 7-methyl G and G (Hallerand Sarnow, Proc. Natl. Acad. Sci. USA 94:8521-8526 (1997)) or between Dand L-tryptophan (supra, Gold et al.). An aptamer can be selected basedon the particular molecule targeted (e.g., aptamer targeting EGFR orother cancer markers). Standard procedures for in vitro selection areknown, such as SELEX experiments, described at Science 249 (4968)505-510 (1990), and Nature (London), 346 (6287) 818-822 (1990) which canbe followed throughout, or with modifications and improvements known inthe art.

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, animal or human that is being sought by theresearcher, veterinarian, medical doctor or other clinician.

By “pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The terms “administration of” and or “administering” should beunderstood to mean providing a composition in a therapeuticallyeffective amount to the individual in need of treatment. Administrationcan be intratumoral or systemic (intravenous) administration.Furthermore, in conjunction with vaccination of recipient with pathogenantigen vaccine (e.g. tetanus toxoid). In addition, in conjunction withagent to deplete or inactivate regulatory T cells (e.g.cyclophosphamide) or myeloid suppressor cells (e.g. gemcitabine). In afurther example, ex vivo treatment of immune cells and tumor cells forgeneration of tumor reactive or pathogen antigen reactive immunecells—for adoptive cellular immunotherapy. Administration can beintradermal or subcutaneous.

Furthermore, administration can be in combination with one or moreadditional therapeutic agents deplete or inactivate regulatory T cells(cyclophosphamide) or myeloid suppressor cells (e.g. gemcitabine). Thepharmaceutical compositions of the invention identified herein areuseful for parenteral, topical, oral, nasal (or otherwise inhaled),rectal, or local administration, such as by aerosol or transdermally,for prophylactic and/or therapeutic treatment of one or more of thepathologies/indications described herein (e.g., cancer, pathogenicinfectious agents, associated conditions thereof). The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. Suitable unit dosage forms,include, but are not limited to powders, tablets, pills, capsules,lozenges, suppositories, patches, nasal sprays, injectables, implantablesustained-release formulations, lipid complexes, etc.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, as it will be understood thatmodifications and variations are encompassed within the spirit and scopeof the instant disclosure.

The following examples are provided to further illustrate theembodiments of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLE 1

Counteracting Tumor Immune Tolerance via Antibody-mediated Depletion ofCD4⁺ Regulatory T Cells Facilitates the Activation of Tumor-ReactiveCD8⁺ T Cells and Enhances the In Vivo Antitumor Efficacy of CytotoxicAnticancer Agents.

Immunogenic death of tumor cells by chemotherapeutic agents can induceCD8⁺ T cell-mediated antitumor immunity. In response to specificchemotherapeutic agents, tumor cells exhibit the rapid translocation ofintracellular calreticulin (CRT) to the cell surface where itsaggregation provides a signal for the recognition and engulfment ofdying tumor cells by antigen presenting dendritic cells (DCs). Treatmentof mouse MB49 or human SW780 bladder cancer cells with doxorubicin, ananthracycline chemotherapeutic agent, induced rapid surface exposure ofCRT that was detected by immunofluorescence cytometry of cells stainedwith Dylight 488-labeled anti-CRT antibody (FIG. 53A). To determinewhether ex vivo treatment with doxorubicin induced an immunogenic deathof tumor cells, either untreated live MB49 cells or an equivalent numberof MB49 cells that were pre-treated in vitro with doxorubicin wereinjected into one flank of syngeneic immunocompetent C57BL/6 mice.Unlike mice injected with live tumor cells, mice injected withdoxorubicin-treated tumor cells exhibited increased production of IFN-γby draining lymph node (DLN) cells in response to in vitro re-challengewith MB49 cell lysates (FIG. 53B). Vaccination with doxorubicin-killedMB49 cells generated a tumor-specific immune response since nocorresponding increase in IFN-γ secretion by DLN cells was observedfollowing in vitro exposure to an irrelevant peptide (Hemagglutinin-HA).Injection of doxorubicin-treated MB49 tumor cells protected mice againsttumor growth upon challenge with untreated live MB49 tumor cellsinjected into the opposite flank. (FIG. 1C). The protection againsttumor growth by vaccination with doxorubicin-treated tumor cells was notobserved in mice that were depleted of CD8⁺ T cells with an anti-CD8antibody before challenge with live tumor cells (FIG. 53C). Theseobservations indicate that ex vivo treatment with chemotherapeuticagents can induce an immunogenic death of tumor cells that generatesCD8⁺ T cell-mediated adaptive antitumor immunity.

Tumor-induced immune tolerance inhibits activation of CD8⁺ T cells inresponse to chemotherapy. To examine whether in vivo treatment withchemotherapeutic agents can activate CD8⁺ T cell-mediated immuneresponses in mice with pre-established tumors, C57BL/6 mice wereinjected with live syngeneic MB49 tumor cells and then administeredintratumoral doxorubicin at various time points following tumorinoculation. In contrast to vaccination of naïve mice withdoxorubicin-killed MB49 cells, in vivo treatment of mice withestablished MB49 tumors at d10 following tumor inoculation failed toinduce a corresponding increase in IFN-γ secretion by DLN cells inresponse to in vitro re-challenge with MB49 cell lysates (FIG. 53B).Whereas treatment with doxorubicin on d3 following tumor inoculation wasable to arrest tumor growth, delayed administration of the same dose ofdoxorubicin on d10 failed to inhibit the progressive growth ofestablished MB49 tumors (FIG. 53D). These results indicate thattumor-induced immune tolerance in the microenvironment of establishedcancers counteracts the activation of adaptive antitumor immunity inresponse to chemotherapy-induced tumor cell death.

Regulatory T cells (Treg) accumulate in the tumor microenvironment andcounteract the ability of chemotherapy to activate CD8⁺ T cell mediatedantitumor immunity. To investigate whether FoxP3⁺ Tregs are involved inenforcing immune tolerance in the tumor microenvironment, we examinedthe percentage of CD4⁺CD25⁺FoxP3⁺ cells (Tregs) among CD4⁺ T lymphocytesin the spleen, draining lymph nodes (DLN), and tumors of immunocompetentC57BL/6 mice at d0 and d14 after tumor inoculation. Whereastumor-bearing mice exhibited only a minor increase in the percentage ofTregs among CD4⁺ T cells in the spleen and DLN at d14 following tumorinoculation, a majority of tumor-infiltrating CD4⁺ T cells at this timewere CD4⁺CD25⁺FoxP3⁺ cells (FIG. 53E). To investigate whether Tregsinfiltrating the tumor microenvironment can suppress the activation ofadaptive antitumor immunity in response to chemotherapy-induced tumorcell death, CD4⁺CD25⁺ cells isolated from tumors and DLN oftumor-bearing mice were adoptively transferred into syngeneic C57BL/6naïve mice before vaccination with doxorubicin-killed MB49 cells. Theadoptive transfer of tumor-infiltrating CD4⁺CD25⁺ cells into naïve miceinhibited the ability of subsequent in vivo vaccination withdoxorubicin-treated MB49 tumor cells to increase production of IFN-γ bydraining lymph node (DLN) cells in response to in vitro re-challengewith MB49 cell lysates (FIG. 53F). Consistent with the ability oftumor-infiltrating CD4⁺CD25⁺ cells to suppress the tumor-specific immuneresponse, the adoptive transfer of these cells counteracted theprotection conferred by vaccination with doxorubicin-treated MB49 cellsagainst tumor growth upon challenge with untreated live MB49 tumor cells(FIG. 53G). These results indicate that the tumor microenvironmentfosters the accumulation of FoxP3⁺ Tregs which counteract the activationof CD8⁺ T cell mediated antitumor immunity in response tochemotherapy-induced tumor cell death.

Inhibition of TGF-β in the tumor microenvironment reducestumor-infiltrating FoxP3⁺ regulatory T cells and enhances the antitumorefficacy chemotherapy. TGF-β induces FoxP3 expression in naïveperipheral CD4⁺CD25⁻FoxP3⁻ T cells and facilitates their conversion into‘adaptive’ FoxP3⁺ Tregs that share the immunosuppressive ability ofnatural FoxP3⁺ Tregs generated in the thymus. Since human cancersfrequently become refractory to the growth-inhibitory effect of TGF-βand acquire an ability to increase expression and secretion of TGF-β, weinvestigated whether this switch enables tumor cells to increase thenumber of adaptive Tregs in the tumor microenvironment. Examination ofserum levels of TGF-β in mice at d0, d14, and d28 following inoculationof live MB49 tumor cells demonstrated that tumor growth resulted in aprogressive increase in the level of serum TGF-β (FIG. 54A). To assessthe precise source of TGF-β in tumor-bearing mice, the total amount ofTGF-β in supernatants of tumor cells or draining lymph node cellsisolated from tumor-bearing mice were measured following ex vivo culturein serum-free medium for 24 h. Measurement of the level of TGF-β/10⁶cells showed that tumor cells were the dominant source of the increasedlevel of TGF-β in tumor-bearing mice (FIG. 54B). In addition to tumorcell-autonomous expression of TGF-β, T cells from tumor-bearing micealso expressed higher levels of TGF-β compared to their counterpartsfrom tumor-free mice (FIG. 54B). To determine whether the elevation ofTGF-β is responsible for the upregulation of Tregs in the tumormicroenvironment, tumor-bearing mice were treated with a solublechimeric protein comprising the extracellular domain of TGFβRII and theFc portion of the murine IgG1 heavy chain (TGFβRII:Fc). This fusionprotein interferes with the binding of TGF-β to endogenous TGFβRII andfunctions as a stable TGF-β antagonist. ELISA assays confirmed theability of TGFβRII:Fc to sequester TGF-β in supernatants of MB49 tumorcells in a concentration-dependent manner (FIG. 54C). At 5 d followinginoculation of MB49 tumor cells, mice were either left untreated ortreated with TGFβRII:Fc (1 μg intratumoral; twice weekly) for 3 weeksfollowed by flow cytometric analyses of intracellular FoxP3 expressionin CD4⁺CD25⁺ T cells infiltrating the tumors. In vivo treatment oftumors with TGFβRII:Fc resulted in a significant decline in FoxP3expression in tumor-infiltrating CD4⁺ T cells (FIG. 54D) and a dramaticreduction of CD4⁺CD25⁺FoxP3⁺ Tregs in tumor tissue (FIG. 54E). Todetermine whether inhibition of TGF-β in the tumor microenvironment canimprove the antitumor efficacy of chemotherapy, MB49 tumor-bearing micewere administered doxorubicin (5 mg/kg i.p. weekly×3) with or withouttwice weekly treatment with TGFβRII:Fc (1 μg intratumoral). In contrastto treatment with either doxorubicin or TGFβRII:Fc alone, combinedtreatment with both agents was able to arrest the growth of MB49 tumors.These results indicate that tumor cell autonomous expression of TGF-β inthe tumor microenvironment induces ‘adaptive’ FoxP3⁺ Tregs and thatcounteracting tumor-induced TGF-β-mediated immune tolerance enhances theantitumor efficacy of chemotherapy.

Anti-CD4 antibody-mediated depletion of CD4⁺ regulatory T cellsfacilitates chemotherapy-induced activation of tumor-reactive CD8⁺ Tcells and enhances the antitumor efficacy of chemotherapy. To determinewhether depletion of CD4⁺ regulatory T cells can improve the antitumorefficacy of chemotherapy by enhancing the activity of CD8⁺ T cells inthe tumor microenvironment, immunocompetent mice bearing syngeneictumors were administered an anti-CD4 antibody (Clone GK1.5) to depleteCD4⁺ T cells or an anti-CD8 antibody (Clone GK2.43) to deplete CD8⁺ Tcells and then treated with specific chemotherapeutic agents. Flowcytometric analyses of peripheral blood mononuclear cells from MB49tumor-bearing mice at d7 following administration of anti-CD4 antibodyor anti-CD8 antibody confirmed the target-specific depletion of eitherCD4⁺ T cells or CD8⁺ T cells, respectively (FIG. 55A). Mice treated withanti-CD4 antibody showed loss of CD4⁺CD25⁺FoxP3⁺ T cells in theperipheral blood as well as among tumor-infiltrating cells (FIGS. 55A,55B). To determine whether antibody-mediated depletion ofCD4⁺CD25⁺FoxP3⁺ cells facilitates chemotherapy-induced activation oftumor-reactive CD8⁺ T cells in the tumor microenvironment, we evaluatedthe expression of IFN-γ in CD8⁺ T cells extracted from the tumor anddraining lymph node of MB49 tumor-bearing mice that were left untreatedor treated with doxorubicin (with or without anti-CD4 antibody). Flowcytometric analyses showed that CD8⁺ T cells from untreated mice did notexpress IFN-γ in response to in vitro re-challenge with MB49 celllysates (FIG. 55C). Whereas IFN-γ⁺CD8⁺ T cells became evident in micetreated with doxorubicin alone, antibody-mediated depletion of CD8⁺ Tcells further enhanced the percentage of tumor-reactive CD8⁺ T cellsthat expressed IFN-γ in doxorubicin-treated animals (FIG. 55C). Todirectly evaluate whether the activation of tumor-reactive CD8⁺ T cellsdetermines the in vivo antitumor efficacy of chemotherapy, we examinedthe effect of antibody-mediated depletion of CD8⁺ T cells or CD4⁺ Tcells on the response of MB49 tumor-bearing mice to systemic treatmentwith doxorubicin (5 mg/kg). Treatment with doxorubicin alone inhibitedthe growth of MB49 tumors but failed to arrest tumor progression.Whereas depletion of CD8⁺ T cells completely impaired the in vivoantitumor efficacy of doxorubicin, depletion of CD4⁺ T cells enhancedthe response to doxorubicin and resulted in tumor regression (FIG. 55D).

Anti-CD4 antibody-mediated depletion of CD4⁺ regulatory T cells augmentsand sustains the antitumor effect of chemotherapy by enabling activationof adaptive antitumor immunity. Whereas tumor cells treated withanthracyclins, such as doxorubicin, are particularly effective ineliciting an antitumor immune response, other chemotherapeutic agentsare less effective in inducing immunogenic tumor cell death. The surfaceexposure of calreticulin is a key determinant of the immunogenicity oftumor cell death in response to chemotherapeutic agents. Compared to theefficient translocation of CRT to the cell surface in response totreatment with doxorubicin (FIG. 1A), treatment of MB49 tumor cells withequitoxic doses of either cisplatin or the combination of cisplatin andgemcitabine was less effective in increasing CRT exposure (FIG. 56A).Whereas tumor-reactive IFN-γ⁺CD8⁺ T cells were evident in tumors of MB49tumor-bearing mice treated with doxorubicin (FIG. 55C), treatment withcisplatin was unable to induce a corresponding elevation of IFN-γexpression in CD8⁺ T cells in response to in vitro re-challenge withMB49 cell lysates (FIG. 56B). To examine whether counteraction ofTreg-mediated immune tolerance enables the activation of antitumorimmunity by cisplatin, immunocompetent tumor-bearing mice were treatedwith cisplatin following depletion of Tregs with anti-CD4 antibody.Antibody-mediated depletion of CD4⁺ T cells enhanced the percentage oftumor-reactive IFN-γ⁺CD8⁺ T cells as well as CD8⁺CD62L⁻ T cells incisplatin-treated animals (FIGS. 56B, 56C). Treatment of MB49tumor-bearing mice with cisplatin partially inhibited tumor growth butfailed to arrest tumor progression. Whereas depletion of CD8⁺ T cellscompletely negated the in vivo antitumor effect of cisplatin, depletionof CD4⁺ T cells enhanced the response to cisplatin and arrested tumorgrowth (FIG. 56D). Although treatment of tumor-bearing mice with thecombination of cisplatin and gemcitabine was also able to arrest tumorgrowth, tumor growth rapidly resumed following termination of therapywith none of the animals (0/8) being tumor-free at d50 following tumorinoculation (FIG. 56E). In contrast, mice depleted of CD4⁺ T cellsexhibited a more sustained response to either single agent orcombination chemotherapy, with 7/16 mice exhibiting complete tumorregression. The complete regression of tumors was attended withestablishment of adaptive antitumor immunity since none of the curedmice (7/7) developed tumors when re-challenged with live MB49 tumorcells in the opposite flank.

Chemotherapy-induced expression of NKG2D ligands on tumor cellscooperates with depletion of CD⁴⁺ regulatory T cells to stimulate CD⁸⁺ Tcell-mediated tumor regression. NKG2D (NK group 2, member D) is alectin-like type II transmembrane stimulatory receptor used by NK cells,γδ-TC^(R+) T cells and αβ-TC^(R+) T cells for immune surveillance oftumors. Expression of mouse and human ligands for NKG2D is upregulatedin transformed epithelial cell lines in response to genotoxic stress orstalled DNA replication, via activation of a DNA damage checkpointpathway initiated by ATM (ataxia telengiectasia, mutated) or ATR (ATM-and Rad3-related) protein kinases. Treatment of CT26 mouse colon cancercells with genotoxic chemotherapeutic agents resulted in upregulation ofmouse NKG2D ligands of the retinoic acid inducible gene family (Rae1)(FIG. 57A). RT-PCR showed that Rae1 mRNA was induced in CT26 cells by2-4 h, reached a plateau after 16-24 h, and began to decline after 48 hof treatment with either the irinotecan or oxaliplatin (FIG. 57A). Flowcytometric analysis demonstrated that cell surface expression of humanNKG2D ligands (MHC-I-related A and B molecules—MICA, MICB) was alsoupregulated on human colorectal cancer cells (HCT116) in response totreatment with irinotecan (FIG. 53B). Isogenic HCT116 cells that differonly in their p53 status demonstrated that p53 is not required foririnotecan-induced upregulation of MICA/B (FIG. 53B). To examine whetherthe induction of NKG2D ligands contributes to the antitumor effect ofchemotherapy in vivo, immunocompetent Balb/C mice inoculated withsyngeneic CT26 tumor cells were treated with irinotecan (50 mg/kg i.p)with or without pre-treatment with an NKG2D blocking antibody (200 μgi.p.). Whereas treatment with irinotecan alone inhibited the growth ofCT26 tumors, the antitumor effect of irinotecan was negated bypre-treatment with the NKG2D blocking antibody (FIG. 1C). Sinceengagement of NKG2D by its ligands provides a costimulatory signal forthe activation of CD⁸⁺ T cells, we investigated whether DNAdamage-induced expression of NKG2D ligands on tumor cells cooperateswith depletion of CD⁴⁺ regulatory T cells to stimulate CD⁺ Tcell-mediated tumor regression. Balb/C mice bearing CT26 tumors wereadministered an anti-CD4 antibody (Clone GK1.5) to deplete CD⁴⁺ T cellsand/or an anti-CD8 antibody (Clone GK2.43) to deplete CD⁺ T cells andthen treated with irinotecan. Flow cytometric analyses confirmed theloss of CD⁴⁺CD2⁵⁺FoxP³⁺ T cells in the spleen and draining lymph node ofmice treated with anti-CD4 antibody (FIG. 57D). Antibody-mediateddepletion of CD⁴⁺ T cells enhanced the percentage of tumor-reactiveIFN-^(γ+)CD⁸⁺ T cells in irinotecan-treated animals (FIG. 57E). Whereastreatment of CT26 tumor-bearing mice with irinotecan only slowed tumorgrowth, depletion of CD⁴⁺ T cells enhanced the response to irinotecanand arrested tumor growth (FIG. 57F). The ability of CD⁴⁺ T celldepletion to augment the antitumor efficacy of irinotecan was mediatedby CD⁸⁺ T cells since antibody-mediated depletion of CD⁸⁺ T cellscompletely negated the in vivo antitumor effect of chemotherapy in CD⁴⁺T cell-depleted mice (FIG. 57F).

These data provide the following insights: (i) activation oftumor-reactive CD8⁺ T cells in response to immunogenic tumor cell deathis a crucial determinant of the antitumor efficacy of chemotherapy invivo; (ii) tumor-induced Tregs impair the antitumor efficacy ofchemotherapy by inhibiting the activation of CD8⁺ T cells in the tumormicroenvironment; and (iii) Counteracting tumor-induced immune tolerancevia antibody-mediated depletion of CD4⁺ regulatory T cells facilitateschemotherapy-induced activation of antitumor immunity with memory,thereby enhancing the antitumor efficacy of chemotherapy; (iv)Strategies to decrease the number or function of CD4+ regulatory T cellsin the tumor microenvironment can increase the activation of CD8+ Tcells and improve the response of tumors to cytotoxic anticancer agents(chemotherapy, tumor-targeted antibodies, targeted therapeutics, kinaseinhibitors) or chemoimmunotherapy (combination of chemotherapeutic agentwith immunotherapeutic agents).

EXAMPLE 2

Exemplary Targeted Immunomodulatory Antibodies and Fusion Proteins

A targeting moiety, including an antibody, can be coupled to animmunomodulatory moiety including a polypeptide derived from theextracellular domain of TGFBR2. Crosslinkers or activating agents forsuch coupling or conjugation are well known in the art. Alternatively,the fusion proteins of the invention can be synthesized usingrecombination DNA technology well known in the art where the codingsequences of various portions of the fusion proteins can be linkedtogether at the nucleic acid level. Subsequently the fusion proteins ofthe invention can be produced using a host cell well known in the art.Examples of targeted immunomodulatory antibodies and fusion proteins areshown in FIGS. 1-33 and briefly described below.

In one embodiment, the present invention provides a molecule including atargeting moiety fused with an immunomodulatory moiety, wherein thetargeting moiety specifically binds to a target molecule, and theimmunomodulatory moiety specifically binds to Transforming growthfactor-beta (TGF-β). SEQ ID NO: 1 provides a fusion protein includinganti-HER2/neu antibody and Transforming growth factor-beta receptor II(TGFβ-RII) Extracellular domain (ECD) (FIG. 2). SEQ ID NO: 2 provides afusion protein including anti-EGFR1 antibody and Transforming growthfactor-beta receptor II (TGFβ-RII) Extracellular domain (ECD) (FIG. 3).SEQ ID NO: 3 provides a fusion protein including anti-CD20 antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD) (FIG. 4). SEQ ID NO: 4 provides a fusion protein includinganti-VEGF antibody and Transforming growth factor-beta receptor II(TGFβ-RII) Extracellular domain (ECD) (FIG. 5). SEQ ID NO: 5 provides afusion protein including anti-human CTLA-4 antibody and Transforminggrowth factor-beta receptor II (TGFβ-RII) Extracellular domain (ECD)(FIG. 6). SEQ ID NO: 6 provides a fusion protein including IL-2, Fc, andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD) (FIG. 7). SEQ ID NO: 7 provides a fusion protein includingTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD), Fc, and IL-2 (FIG. 7). SEQ ID NO: 8 provides a fusionprotein including anti-CD25 antibody and Transforming growth factor-betareceptor II (TGFβ-RII) Extracellular domain (ECD) (FIG. 8A). SEQ ID NO:9 provides a fusion protein including anti-CD25 antibody andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ECD) (FIG. 8B). SEQ ID NO: 10 provides a fusion proteinincluding anti-CD4 antibody and Transforming growth factor-beta receptorII (TGFβ-RII) Extracellular domain (ECD) (FIG. 9). SEQ ID NO: 11provides a fusion protein including PD-1 Ectodomain, Fc, andTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ectodomain) (PD-1 ectodomain+Fc+TGFβRII ectodomain; FIG. 10).SEQ ID NO: 12 provides a fusion protein including Transforming growthfactor-beta receptor II (TGFβ-RII) Extracellular domain (ectodomain),Fc, and PD-1 Ectodomain (TGFβRII ectodomain+Fc+PD-1 ectodomain; FIG.10). SEQ ID NO: 13 provides a fusion protein including RANK Ectodomain,Fc, and Transforming growth factor-beta receptor II (TGFβ-RII)Extracellular domain (ectodomain) (RANK ectodomain+Fc+TGFβRIIectodomain; FIG. 11). SEQ ID NO: 14 provides a fusion protein includingTransforming growth factor-beta receptor II (TGFβ-RII) Extracellulardomain (ectodomain), Fc, and RANK Ectodomain (TGFβRII ectodomain+Fc+RANKectodomain; FIG. 11).

In another embodiment, the present invention provides a moleculeincluding a targeting moiety fused with an immunomodulatory moiety,wherein the targeting moiety specifically binds to a target molecule,and the immunomodulatory moiety is a molecule that specifically binds toProgrammed Death-1 ligand 1 (PD-L1 or B7-H1) or Programmed Death-1ligand 2 (PD-L2 or B7-DC). SEQ ID NO: 15 provides a fusion proteinincluding anti-HER2/neu antibody and PD-1 Ectodomain (FIG. 13). SEQ IDNO: 16 provides a fusion protein including anti-EGFR1 antibody and PD-1Ectodomain (FIG. 14). SEQ ID NO: 17 provides a fusion protein includinganti-CD20 antibody and PD-1 Ectodomain (FIG. 15). SEQ ID NO: 18 providesa fusion protein including anti-VEGF antibody and PD-1 Ectodomain (FIG.16). SEQ ID NO: 19 provides a fusion protein including anti-human CTLA-4antibody and PD-1 Ectodomain (FIG. 17). SEQ ID NO: 20 provides a fusionprotein including anti-CD25 antibody and PD-1 Ectodomain (FIG. 18A). SEQID NO: 21 provides a fusion protein including anti-CD25 antibody andPD-1 Ectodomain (FIG. 18B). SEQ ID NO: 22 provides a fusion proteinincluding IL-2, Fc, and PD-1 ectodomain (IL-2+Fc+PD-1 ectodomain; FIG.19). SEQ ID NO: 23 provides a fusion protein including PD-1 ectodomain,Fc, and IL-2 (PD-1 ectodomain+Fc+IL-2; FIG. 19). SEQ ID NO: 24 providesa fusion protein including anti-CD4 antibody and PD-1 Ectodomain (FIG.20). SEQ ID NO: 25 provides a fusion protein including RANK Ectodomain,Fc, and PD-1 ectodomain (RANK ectodomain+Fc+PD-1 ectodomain; FIG. 21).SEQ ID NO: 26 provides a fusion protein including PD-1 ectodomain, Fc,and RANK Ectodomain (PD-1 ectodomain+Fc+RANK ectodomain; FIG. 21).

In another embodiment, the present invention provides a moleculeincluding a targeting moiety fused with an immunomodulatory moiety,wherein the targeting moiety specifically binds to a target molecule,and the immunomodulatory moiety is a molecule that specifically binds toReceptor activator of NF-kB ligand (RANKL). SEQ ID NO: 27 provides afusion protein including anti-HER2/neu antibody and RANK Ectodomain(FIG. 23). SEQ ID NO: 28 provides a fusion protein including anti-EGFR1antibody and RANK Ectodomain (FIG. 24). SEQ ID NO: 29 provides a fusionprotein including anti-CD20 antibody and RANK Ectodomain (FIG. 25). SEQID NO: 30 provides a fusion protein including anti-VEGF antibody andRANK Ectodomain (FIG. 26). SEQ ID NO: 31 provides a fusion proteinincluding anti-human CTLA-4 antibody and RANK Ectodomain (FIG. 27). SEQID NO: 32 provides a fusion protein including anti-CD25 antibody andRANK Ectodomain (FIG. 28A). SEQ ID NO: 33 provides a fusion proteinincluding anti-CD25 antibody and RANK Ectodomain (FIG. 28B). SEQ ID NO:34 provides a fusion protein including IL-2, Fc, and RANK ectodomain(IL-2+Fc+RANK ectodomain; FIG. 29). SEQ ID NO: 35 provides a fusionprotein including RANK ectodomain, Fc, and IL-2 (RANKectodomain+Fc+IL-2; FIG. 29). SEQ ID NO: 36 provides a fusion proteinincluding anti-CD4 antibody and RANK Ectodomain (FIG. 30).

In another embodiment, the present invention provides a moleculeincluding a targeting moiety fused with an immunomodulatory moiety,wherein the targeting moiety specifically binds to a target molecule,and the immunomodulatory moiety includes a molecule that specificallybinds to Programmed death-1 (PD-1). SEQ ID NO: 37 provides a fusionprotein including anti-tumor necrosis factor (TNFα) antibody and PD-1ligand 1 (FIG. 32). SEQ ID NO: 38 provides a fusion protein includingTNFR2 Extracellular ligand binding domain, Fc, and PD-1 ligand: (TNFR2ECD+IgG Cγ1+PD-L1; FIG. 33). SEQ ID NO: 39 provides a fusion proteinincluding PD-1 ligand, Fc, and TNFR2 Extracellular ligand bindingdomain: (PD-L1+IgG Cγ1-TNFR2 ECD; FIG. 33). SEQ ID NO: 40 provides afusion protein including anti-CD20 antibody and PD-1 ligand 1 (PD-L1)(FIG. 34). SEQ ID NO: 41 provides a fusion protein including anti-CD25antibody and PD-1 ligand 1 (PD-L1) (FIG. 35A). SEQ ID NO: 42 provides afusion protein including anti-CD25 antibody and PD-1 ligand 1 (PD-L1)(FIG. 35B). SEQ ID NO: 43 provides a fusion protein including PD-1ligand 1 (PD-L1), Fc, and IL-2 (PD-L1-Fc-IL2; FIG. 36). SEQ ID NO: 44provides a fusion protein including IL-2, Fc, and PD-1 ligand 1 (PD-L1)(IL-2-Fc-PD-L1; FIG. 36). SEQ ID NO: 45 provides a fusion proteinincluding anti-CD4 antibody and PD-1 ligand 1 (PD-L1) (FIG. 37). SEQ IDNO: 46 provides a fusion protein including the extracellular domain ofCTLA-4, Immunoglobulin Fc (IgG Cγ1), and a sequence from PD-1 ligand(PD-L1) (Oncostatin M signal peptide+CTLA-4 ECD+IgG Cγ1+PD-L1; FIG. 38).SEQ ID NO: 47 provides a fusion protein including the extracellulardomain of PD-1 ligand (PD-L1), immunoglobulin Fc (IgG Cγ1), and asequence from the extracellular domain of CTLA-4: (PD-L1+IgG Cγ1+CTLA-4ECD; FIG. 38). SEQ ID NO: 48 provides a fusion protein includingTransforming growth factor-β (TGF-β), immunoglobulin Fc (IgG Cγ1), and asequence from PD-1 ligand 1 (PD-L1) (TGFβ-1+Fc+PD-L1; FIG. 39). SEQ IDNO: 49 provides a fusion protein including a sequence from PD-1 ligand 1(PD-L1), immunoglobulin Fc (IgG Cγ1), and Transforming growth factorbeta (TGF-□β) (PD-L1+Fc+TGFβ-1; FIG. 39).

In another embodiment, the present invention provides a moleculeincluding a targeting moiety fused with an immunomodulatory moiety,wherein the targeting moiety specifically binds to a target molecule,and the immunomodulatory moiety includes a molecule that specificallybinds to Transforming growth factor-beta receptor (TGF-βR). SEQ ID NO:50 provides a fusion protein including an antibody that binds TNF-α, anda sequence from Transforming growth factor-β (TGF-β) (FIG. 41). SEQ IDNO: 51 provides a fusion protein including TNFR2 Extracellular ligandbinding domain, Fc, and a sequence from Transforming growth factor-β(TGF-β) (TNFR2 ECD+IgG Cγ1+TGF-β; FIG. 42). SEQ ID NO: 52 provides afusion protein including a sequence from Transforming growth factor-β(TGF-β), Fc, and TNFR2 Extracellular ligand binding domain: (TGF-β+IgGCγ1+TNFR2 ECD; FIG. 42). SEQ ID NO: 53 provides a fusion proteinincluding anti-CD20 antibody and a sequence from Transforming growthfactor-β (TGF-β) (FIG. 43). SEQ ID NO: 54 provides a fusion proteinincluding anti-CD25 antibody and a sequence from transforming growthfactor-β (TGF-β) (FIG. 44A). SEQ ID NO: 55 provides a fusion proteinincluding anti-CD25 antibody and a sequence from transforming growthfactor-β (TGF-β) (FIG. 44B). SEQ ID NO: 56 provides a fusion proteinincluding a sequence from Transforming growth factor-β (TGF-β), Fc, andIL-2 (TGF-β+Fc+IL-2; FIG. 45). SEQ ID NO: 57 provides a fusion proteinincluding IL-2, Fc, and Transforming growth factor-β(TGF-β)(IL-2+Fc+TGF-β; FIG. 45). SEQ ID NO: 58 provides a fusion proteinincluding anti-CD4 antibody and a sequence from transforming growthfactor-β (TGF-β) (FIG. 46). SEQ ID NO: 59 provides a fusion proteinincluding the extracellular domain of CTLA-4, immunoglobulin Fc (IgGCγ1), and a sequence from a sequence from transforming growth factor-β(TGF-β) (Oncostatin M signal peptide+CTLA-4 ECD+IgG Cγ1+TGF-β1; FIG.47). SEQ ID NO: 60 provides a fusion protein including a sequence fromTransforming growth factor-β (TGF-β), immunoglobulin Fc (IgG Cγ1), and asequence from the extracellular domain of CTLA-4: (TGF-β1+IgG Cγ1+CTLA-4ECD) (FIG. 47).

In another embodiment, the present invention provides a moleculeincluding a targeting moiety fused with an immunomodulatory moiety,wherein the targeting moiety specifically binds to a target molecule,and the immunomodulatory moiety is a molecule that specifically binds toReceptor activator of NF-kB ligand (RANKL). SEQ ID NO: 61 provides afusion protein including an antibody that binds TNF-α, and a sequencefrom RANK Ectodomain (FIG. 48). SEQ ID NO: 62 provides a fusion proteinincluding TNFR2 Extracellular ligand binding domain, Fc, and a sequencefrom RANK Ectodomain (TNFR2 ECD+IgG Cγ1+RANK Ectodomain; FIG. 49). SEQID NO: 63 provides a fusion protein including a sequence from RANKEctodomain, Fc, and TNFR2 Extracellular ligand binding domain: (RANKEctodomain+IgG Cγ1+TNFR2 ECD; FIG. 49). SEQ ID NO: 64 provides a fusionprotein including the extracellular domain of CTLA-4, immunoglobulin Fc(IgG Cγ1), and a sequence from a sequence from RANK Ectodomain(Oncostatin M signal peptide+CTLA-4 ECD+IgG Cγ1+RANK Ectodomain; FIG.50). SEQ ID NO: 65 provides a fusion protein including a sequence fromRANK Ectodomain, immunoglobulin Fc (IgG Cγ1), and a sequence from theextracellular domain of CTLA-4: (RANK Ectodomain+IgG Cγ1+CTLA-4 ECD)(FIG. 50). SEQ ID NO: 66 provides a fusion protein including a sequencefrom transforming growth factor-β (TGF-β), immunoglobulin Fc region (IgGCγ1), and an extracellular ligand-binding domain or ectodomain ofReceptor activator of nuclear factor-KB (RANK) (TGF-β+IgG Cγ1+RANKEctodomain; FIG. 51). SEQ ID NO: 67 provides a fusion protein includinga sequence from RANK Ectodomain, immunoglobulin Fc (IgG Cγ1), and asequence from transforming growth factor-β (TGF-β): (RANK Ectodomain+IgGCγ1+TGF-β) (FIG. 51). SEQ ID NO: 68 provides a fusion protein includinga sequence from Programmed death-1 ligand 1 (PD-L1), immunoglobulin Fcregion (IgG Cγ1), and an extracellular ligand-binding domain orectodomain of Receptor activator of nuclear factor-κB (RANK) (PD-L1+IgGCγ1+RANK Ectodomain; FIG. 52). SEQ ID NO: 69 provides a fusion proteinincluding a sequence from RANK Ectodomain, immunoglobulin Fc (IgG Cγ1),and a sequence from Programmed death-1 ligand 1 (PD-L1): (RANKEctodomain+IgG Cγ1+PD-L1) (FIG. 52).

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. An isolated molecule comprising: (a) a targetingmoiety that is a polypeptide that specifically binds Interleukin-6Receptor (IL6R); and (b) an immunomodulatory moiety comprising an aminoacid sequence of the extracellular domain of Transforming growthfactor-beta receptor (TGFβR) or a fragment thereof, wherein theimmunomodulatory moiety comprises an amino acid sequence of theextracellular domain of Transforming growth factor-beta receptorselected from the group consisting of SEQ ID NO: 79-91 or fragmentthereof.
 2. The molecule of claim 1, wherein the targeting moietycomprises an antibody, antibody fragment, antigen-binding domain of animmunoglobulin, single chain variable fragment (scFv), Fc-containingpolypeptide, or fusion protein that specifically binds IL6R or afragment thereof.
 3. The molecule of claim 2, wherein the targetingmoiety comprises the amino acid sequence corresponding to the heavychain or light chain of an antibody that specifically binds IL6R.
 4. Themolecule of claim 1, wherein the immunomodulatory moiety comprises anamino acid sequence of the extracellular domain of Transforming growthfactor-beta receptor II (TGFβRII) or a binding fragment thereof.
 5. Themolecule of claim 4, wherein the immunomodulatory moiety comprises theamino acid sequence corresponding to SEQ ID NO: 87 or a binding fragmentthereof.
 6. The molecule of claim 1, wherein said immunomodulatorymoiety is fused to the heavy chain or light chain of said targetingmoiety comprising an antibody or to the C-terminus or N-terminus of saidtargeting moiety.
 7. The molecule of claim 1, wherein the targetingmoiety is fused to the immunomodulatory moiety via a linker.
 8. Themolecule of claim 1, wherein the targeting moiety is an Fc-containingpolypeptide further comprising a linker at the CH3 region of the Fc. 9.The molecule of claim 8, wherein the linker is (GGGGS)_(n) (SEQ ID NO:123) and wherein n is 1, 2, 3, 4, 5, 6, 7 or
 8. 10. The isolatedmolecule of claim 9, wherein the linker comprises the amino acidsequence GGGGSGGGGSGGGGS (SEQ ID NO: 104).